Antioxidant Extraction from Mustard ( Brassica juncea ) Seed Meal Using...

E:FoodEngineering&PhysicalProperties

Antioxidant Extraction from Mustard(Brassica juncea) Seed Meal UsingHigh-Intensity UltrasoundJeremiah Dubie, Aaron Stancik, Matthew Morra, and Caleb Nindo

Abstract: Brassicaceae oilseeds provide feedstocks for the biofuels industry, but value-added coproducts are necessaryto supply financial incentives for increased production. Our objective was to use high-intensity ultrasound to optimizeextraction of antioxidants from mustard (Brassica juncea) seed meal. The ultrasound-assisted extraction (UAE) variablesincluded temperature, solvent-to-material ratio, sonication duration, and EtOH concentration. Extracts were analyzedfor total phenolics content (TPC), antioxidant activity, and sinapine content. Conventional extraction using water and70% EtOH (v/v) at 80 ◦C for 3×30 min yielded 7.83 ± 0.07 and 8.81 ± 0.17 mg sinapic acid equivalents (SAE)/gmeal, respectively. UAE extraction at 40 ◦C for 30 min yielded similar phenolics content (8.85 ± 0.33 mg SAE/g meal)as conventional hot ethanolic extraction, but required less time and lower temperature. The highest TPC (13.79 ± 0.38mg SAE/g meal) was in the 7-d aqueous extracts. Sonicated solutions of pure sinapine and sinapic acid showed 1st-orderreaction kinetics with greater degradation of isolated compounds than those present in extracts. Sinapine contained inextracts showed insignificant (P < 0.05) degradation after 30 min of sonication. Our research indicates that ultrasoundtreatment can assist the extraction of antioxidants from B. juncea meal by reducing both the temperature and timerequirement without significant degradation of the primary antioxidants present.

Keywords: antioxidant, DPPH, oilseed meals, Sinapic acid, ultrasound

Practical Application: Brassicaceae seed meals contain residual compounds with antioxidant and antimicrobial propertiesthat may be incorporated in various food products to extend their shelf life. Ultrasound-assisted extraction can potentiallyenhance the extraction of these compounds for the development of value-added products.

IntroductionBrassicaceae oilseed crops, including rapeseed (Brassica napus)

and mustard (Brassica juncea and Sinapis alba), are important becauseof their potential to support the growing biofuels industry. Anincreased financial incentive to grow these crops can be achievedthrough the extraction of value-added coproducts from the seedmeal remaining after oil extraction. Antioxidants are importantin biological systems because of their ability to retard degradationcaused by oxidation. Synthetic antioxidants are very effective, butcan have negative health effects, such as potential pathological,enzyme, and lipid alterations (Branen 1975). Therefore, the use ofnatural antioxidants from plant extracts in food products has beengaining popularity (Porkorny 2007). Extracts of Brassicaceae seedmeals (BSMs) (rapeseed and mustard) have potent antioxidantactivity resulting primarily from phenolic compounds (Kozlowskaand others 1983; Shahidi and others 1994; Wanasundara andothers 1995). The meals contain bioactive phenolic compounds inthe form of 3,5-dimethoxy-4-hydroxycinnamic acid (sinapic acid)derivatives, which have shown stronger antioxidant activity (AA)than many artificial and natural antioxidants (Wanasundara and

MS 20121479 Submitted 10/29/2012, Accepted 1/19/2013. Authors Dubie andNindo are with School of Food Science, Univ. of Idaho, Moscow ID 83844, U.S.A.Authors Stancik and Morra are with Plant, Soil and Entomological Sciences, Univ.of Idaho, Moscow ID 83844, U.S.A. Direct inquiries to author Nindo (E-mail:[email protected]).

Shahidi 1994; Rice-Evans and others 1996). BSM and extractsfrom the same materials have been used in ground meat to reducelipid oxidation, extend shelf life, and preserve quality (Saleemi andothers 1993; Brettonett and others 2010; Lara-Lledo and others2012).

Novel extraction techniques that improve extraction efficiencyincluding the application of high-intensity, low-frequency ultra-sound that is based on physical effects of cavitation are continuallybeing developed (Kentish and Ashokkumar 2011). Ultrasound canincrease cell wall destruction, cause leakage of cellular material,enhance the penetration of solvent into plant cells, facilitate hy-dration and swelling, and improve mass transfer (Vinatoru 2001;Vilkhu and others 2008). These phenomena can increase extrac-tion of antioxidants, while significantly reducing extraction time,thus improving overall efficiency (Mason and others 1996; Albuand others 2004). Ultrasound-assisted extraction (UAE) is versatileand can be used both on small and large scales (Vinatoru 2001).

Despite the known beneficial effects of ultrasound for the ex-traction of bioactive compounds, the potential negative effectsof ultrasound on the compounds of interest have not been ade-quately investigated (Soria and Villamiel 2010). The violent col-lapses that occur during transient cavitation can generate extremelocalized temperatures and pressures exceeding 5000 K and 500bar (Hoffman and others 1996; Thompson and Doraiswamy 1999;Esclapez and others 2011). Those conditions can cause a numberof chemical changes to occur within the cavitation bubble and inthe surrounding liquid, including the formation of free radicals

C© 2013 Institute of Food Technologists R©

E542 Journal of Food Science � Vol. 78, Nr. 4, 2013 doi: 10.1111/1750-3841.12085Further reproduction without permission is prohibited

E:Fo

odEn

ginee

ring&

Phys

icalP

ropert

ies

Antioxidant extraction from mustard (Brassica juncea) seed meal . . .

(Knorr and others 2004) and degradation of bioactive compoundsthrough oxidation by hydroxyl radicals, pyrolysis, and supercriticalwater oxidation (Hoffman and others 1996).

Extraction of phenolics from BSM has conventionally been ac-complished through the use of organic solvents, particularly alco-hols, and high temperatures over long extraction times (Shahidiand others 1994; Wanasundara and Shahidi 2005). Conventionalextraction parameters usually involve the use of 70% or 80% EtOHat 80 ◦C. Meal is extracted for 30 min and filtered, and the solidresidue is extracted 2 additional times under the same conditions.

Ultrasound treatment can improve the extraction of variouscompounds including antioxidants and antimicrobials from BSM(Lin and others 2000; Wang and others 2011). However, additionalresearch is required to maximize extraction yield while minimiz-ing possible degradation caused by the sonochemical effects oftransient cavitation. Our objectives were to optimize the yield ofantioxidants extracted from BSM using high-intensity ultrasoundand to quantify the effect of sonication on the degradation of theextracted phenolic antioxidants.

Methods and Materials

MaterialsAll experiments were performed using meal remaining after

cold pressing of B. juncea “Pacific Gold” (Brown and others1998), S. alba “IdaGold” (Brown and others 2004), and Bras-sica napus seeds in a press on the Univ. of Idaho campus (Moscow,Idaho, U.S.A.). Approximately 90% of the oil was extracted fromthe seeds using a mechanical seed crusher (Peterson and others1983; Borek and Morra 2005). The meal obtained after press-ing had water content between 4% and 5% on the wet weightbasis and consisted of irregularly shaped flakes that were approx-imately 1 mm in thickness and 1 to 3 cm in length (Hendrixand others 2012). The standard compounds sinapic acid ≥98%,2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl free radical (DPPH),2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammo-nium salt (ABTS) ≥ 99.0%, and Folin Ciocalteu’s phenol reagentwere obtained from Sigma-Aldrich (St. Louis, Mo., U.S.A.).

Conventional extractionB. juncea seed meal was extracted with either 70% ethanol or

deionized water according to procedures outlined by Shahidi andothers (1994) and Hassas-Roudsari and others (2009). Six grams ofmeal were placed in a 200-mL Erlenmeyer flask to which 120 mLof solvent was added. The mixture was heated with agitation in awater bath maintained at 80 ◦C for 30 min and filtered throughWhatman nr 2 filter paper. The solid residue was extracted 2additional times under the same conditions. The extracts werecombined and stored at 4 ◦C until analysis. The BSM was alsoextracted in triplicate with either 70% ethanol or deionized waterfor 24 h or 7 d with agitation at room temperature (22 ± 1 ◦C).

UAEB. juncea meal was subjected to low-frequency, high-intensity

ultrasound treatment in a batch system. Parameters were variedindividually (with all others held constant) to determine their ef-fects on the extraction of antioxidants from mustard meal. B. junceameal was extracted with either 70% ethanol or deionized waterat a solvent-to-material ratio of 50 : 1. The extraction solution(120 mL) was subjected to 30 min of 20 kHz and 0.5 W/mLultrasound treatments (Branson Sonifier model S-450A, 400W,

Danbury, Conn., U.S.A.) at 20 ◦C. The titanium probe tip of thesonotrode horn had a length of 10 cm with a diameter of 1.9 cmand was inserted 2 cm into the reaction solution. Parameters werevaried individually in a 4+6+6+5 design as follows: temperature(20, 40, 60, and 75 ◦C), solvent-to-material ratio (10 : 1, 20 : 1,30 : 1, 40 : 1, 50 : 1, and 60 : 1), duration (10, 20, 30, 40, 50, and60 min), and concentration of ethanol in solvent (0%, 30%, 50%,70%, and 95%). Each temperature condition was held maintainedwithin ±1 ◦C using a jacketed stainless steel reaction chamberplaced inside a plastic jacket with water coolant circulating froma refrigerated bath (Bath & circulator model 2095, Forma Scien-tific, Marieta, Ohio, U.S.A.). Temperature was monitored usingthermocouples and a data logger (21X Micrologger, CampbellScientific, Logan, Utah, U.S.A.).

The acoustic power delivered to the reaction solution was deter-mined by the calorimetric method (Contamine and others 1995).The acoustic power density (APD), which represents the intensityof the ultrasonic treatment and is inversely related to the volume(V) of the reaction solution, was determined from the rise in tem-perature of the reaction solution according to Eq. (1) and (2).

AP D = PV

[Wml

](1)

The acoustic power (P) delivered via cavitation was calculatedby the rate of temperature rise (dT/dt), the mass (m), and the heatcapacity (cp) of the solution.

P = mc p

(d Td t

)[W] (2)

Under our experimental conditions, power was determined tobe 60 W and APD was 0.5 W/mL.

After sonication, extraction solutions were filtered throughWhatman nr 2 filter paper and stored at 4 ◦C until analysis.

Total phenolics and antioxidant assaysAll samples were analyzed for total phenolics content (TPC) us-

ing Folin Ciocalteu’s phenol reagent and total antioxidant activity(AA) using DPPH and ABTS assays. Results were compared withconventional hot-solvent extraction. Extracts were assayed usingsinapic acid as the standard and results are given as mg sinapic acidequivalents/g meal (mg SAE/g meal). The primary antioxidants inBSM are sinapic acid derivatives; thus, sinapic acid was consideredas a suitable standard to quantify the antioxidant capacity of theextracts.

The TPC of extracts was determined according to the proce-dure outlined by Amarowicz and others (2004). Fifty microliters ofextract or standard (sinapic acid dissolved in ethanol) were addedto 4.5 mL polystyrene cuvettes containing 2000 µL deionizedwater and 50 µL Folin Ciocalteu’s phenol reagent and vortexedfor 15 s. The contents were allowed to stand for 3 min at roomtemperature before the addition of 300 µL saturated sodium car-bonate solution and a final vortexing for 15 s. These were allowedto stand for another 60 min at room temperature in the dark be-fore recording the absorbance at 725 nm (Lambda 35 UV/VisSpectrophotometer, Perkin Elmer, Akron, Ohio) using deionizedwater as the reference.

The DPPH antioxidant activity of extracts was determined bytheir ability to reduce the DPPH radical (Naczk and others 2005).

Vol. 78, Nr. 4, 2013 � Journal of Food Science E543



The DPPH solution was prepared by dissolving 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl in 95% ethanol to an absorbance of0.70 ± 0.10. Fifteen microliters of extract or standard (sinapic aciddissolved in ethanol) were added to cuvettes containing 2000 µLDPPH solution and vortexed for 15 s. The contents were allowedto stand at room temperature for 10 min before recording theabsorbance at 517 nm using 95% ethanol as the reference.

The ABTS antioxidant activity was determined similarly ac-cording to the procedure outlined by Re and others (1999).Briefly, 38.4 mg of ABTS and 6.6 mg potassium peroxydisul-fate were dissolved in 10 mL deionized water and allowed to standat room temperature in the dark for 16 h. Following incubation,95% EtOH was added to the solution until the absorbance reached0.70 ± 0.10. Ten microliters of extract or standard (sinapic aciddissolved in ethanol) were added to cuvettes containing 2000 µLABTS solution and vortexed for 15 s. The contents were allowedto stand at room temperature for 10 min before recording theabsorbance at 734 nm using 95% ethanol as the reference solution.

Extraction and degradation kineticsSinapine is commonly referred to as the dominant phenolic

compound in BSM (Bouchereau and others 1991; Engels andothers 2012). Samples (0.5 mL) were taken every 10 min up to60 min and analyzed by HPLC to determine the kinetics of sinap-ine extraction from B. juncea meal during sonication with thetemperature held constant at either 20 ± 1 or 75 ± 1 ◦C.

Solutions containing 200 mg/L sinapine, 200 mg/L sinapic acid,or a combination of both were also sonicated to investigate theeffect of high-intensity ultrasound on the degradation of sinapineand sinapic acid. A conventional hot ethanolic extract was filteredthrough a Whatman 0.45-µm PES filter and sonicated at 20 ◦C,after which samples were taken every 10 min up to 60 min andanalyzed by HPLC. This sequence was compared to one usingan extract spiked with sinapic acid and pure solutions of eithersinapine or sinapic acid to investigate possible effects of othercompounds in the reaction solution.

Degradation of phenolic compounds has been found to fol-low 1st-order kinetics (Emery and others 2003). Degradation ofsinapine and sinapic acid was modeled according to the exponen-tial decay equation (Eq. 3), where Co is the initial concentration,Ct is the concentration at the time of sample (t), and k is the rateconstant (1/s). Experiments were conducted in duplicate.

Ct

Co= e−kt (3)

Sinapine thiocyanate was purified according to the method ofMailer and others (2008). B. juncea meal was extracted with 95%ethanol using a Soxhlet apparatus for 4 h. The solution was filteredthrough a 0.45-µm filter membrane and concentrated using arotary evaporator (40 ◦C). The extract was diluted with deionizedwater to which potassium thiocyanate was added (3% w/w finalconcentration), after which the solution was stored at 4 ◦C for 48 h.The sinapine thiocyanate crystals were recovered by centrifugationand decanting. The crystals were then dissolved in 95% ethanoland stored at 4 ◦C for 24 h.

HPLC analysisExtracts were analyzed using an Agilent 1200 HPLC equipped

with an online degasser, binary pump, autosampler, thermostatedcolumn compartment, and diode array detector (Agilent Tech-

Table 1–Effect of conventional extraction methods on TPC andantioxidant activity as measured in extracts from B. juncea seedmeal.a

Antioxidant activity(mg SAE/g meal)

Extraction Total phenolicsmethod (mg SAE/g meal) DPPH assay ABTS assay

Hot waterb 7.83 ± 0.07d 5.71 ± 0.25c 5.72 ± 0.10cd24 h waterc 11.56 ± 0.16b 4.59 ± 0.13d 6.94 ± 0.20b7 d waterc 13.79 ± 0.38a 4.77 ± 0.22d 8.03 ± 0.29aHot EtOHd 8.81 ± 0.17c 7.34 ± 0.37a 5.98 ± 0.22c24 h EtOHe 8.00 ± 0.18d 6.51 ± 0.19b 5.53 ± 0.20de7 d EtOHe 8.53 ± 0.17c 5.72 ± 0.20c 5.31 ± 0.14e

aValues are means of at least 3 measurements ± standard deviation. Meal moisturecontent was between 4% and 5% (wet basis). Means within a column followed by thesame letter are not significantly different (P < 0.05).bHot water extraction at 80 ◦C for 3×30 min.cLong-duration water extraction at 25 ◦C for 24 h or 7 d.dHot 70% EtOH (v/v) extraction at 80 ◦C for 3×30 min.eLong-duration 70% EtOH (v/v) extraction at 25 ◦C for 24 h or 7 d.

nologies, Inc., Santa Clara, Calif., U.S.A.). Separation was per-formed on a 250 × 2.00 mm, 5 µm, 125 A Aqua C18 column(Phenomenex, Torrance, Calif.). Gradient elution was performedusing water with 0.1% formic acid as solvent A and methanol with0.1% formic acid as solvent B. The gradient began with 20% B at0 min, increasing to 80% B at 15 min, and decreasing to 20% Bat 20 min with a postrun of 5 min. Solvents and extract sampleswere filtered through a 0.45-µm filter membrane. The columnwas maintained at 25 ◦C and the flow rate was 250 µL/min.Chromatograms were acquired at 330 nm and data were analyzedusing ChemStation software (Agilent Technologies, Inc.). Peakswere labeled according to their relative retention times comparedto those of reference standards.

Statistical analysisEach UAE treatment condition was tested in triplicate and each

assay was performed in triplicate for each sample. HPLC analysiswas performed in duplicate. One-way analysis of variance andBonferroni t-tests were performed to assess the effects of extractionconditions on the yield of total phenolics and antioxidant activityof extracts (Statistical Analysis Software, SAS/STAT version 9.2).The same software was used to calculate correlation coefficientsamong the 3 assays.

Results and Discussion

Conventional extractionThe results of conventional extraction performed on B. juncea

seed meal are presented in Table 1. The TPC of the hot ethanolicextract is consistent with the range of levels measured in othervarieties of B. juncea (Mailer and others 2008; Khattab and others2010).

The highest TPC and ABTS antioxidant activities were ob-served in the 24-h and 7-d aqueous extracts. In contrast, the low-est DPPH antioxidant activities were measured in these same 24-hand 7-d aqueous extracts. The highest DPPH antioxidant activi-ties occurred in hot ethanol extracts, whereas ABTS antioxidantactivities in hot ethanol extracts were equivalent to those obtainedin hot water extracts. All 3 ethanol extracts yielded lower phenolicconcentrations and ABTS antioxidant activities than their respec-tive 24-h and 7-d aqueous extracts. The opposite was true forDPPH in that ethanol extracts yielded higher activities than 24-h

E544 Journal of Food Science � Vol. 78, Nr. 4, 2013

E:Fo

odEn

ginee

ring&

Phys

icalP

ropert

ies


Table 2–Effect of extraction temperature on TPC and antioxi-dant activity as measured in UAE extracts from B. juncea seedmeal (70% EtOH, 60 W, 120 mL, 50 : 1 solvent, 30 min).a

Antioxidant activityExtraction (mg SAE/g meal)temperature Total phenolics(◦C) (mg SAE/g meal) DPPH assay ABTS assay

20 8.38 ± 0.28c 6.57 ± 0.24c 5.47 ± 0.07c40 8.85 ± 0.33bc 6.99 ± 0.15cb 5.71 ± 0.18c60 9.33 ± 0.44b 7.52 ± 0.09ba 6.37 ± 0.39b75 9.97 ± 0.36a 7.78 ± 0.13a 7.71 ± 0.28a

aValues are means of at least 3 measurements ± standard deviation. Meal moisturecontent was between 4% and 5% (wet basis). Means within a column followed by thesame letter are not significantly different (P < 0.05).

Table 3–Effects of sonication duration on TPC and antioxidantactivity as measured in UAE extracts from B. juncea seed meal(20 ◦C, 70% EtOH, 60 W, 120 mL, 50 : 1 solvent).a

Antioxidant activitySonication (mg SAE/g meal)duration Total phenolics(min) (mg SAE/g meal) DPPH assay ABTS assay

10 6.27 ± 0.37c 5.83 ± 0.17b 4.14 ± 0.27b20 7.24 ± 0.42b 6.20 ± 0.30ab 5.36 ± 0.20a30 8.38 ± 0.28a 6.57 ± 0.24ab 5.47 ± 0.07a40 8.38 ± 0.39a 6.66 ± 0.22a 5.51 ± 0.11a50 8.36 ± 0.22a 6.72 ± 0.18a 5.44 ± 0.16a60 8.14 ± 0.69a 6.44 ± 0.24a 5.46 ± 0.18a


and 7-d aqueous extracts. These phenomena suggest that there aredifferent compounds present in mustard seed meal extracts thatcontribute to the antioxidant activity and these compounds havedifferent solubilities. Our results also suggest that these compoundshave different antioxidant behavior based on the lack of correlationbetween the DPPH and ABTS assays. In addition, it appears thatthere are water-soluble phenolic compounds that do not providemuch AA compared to those extracted with alcohol.

UAEUAE of antioxidants from B. juncea seed meal was studied by

varying several parameters including temperature, sonication dura-tion, EtOH concentration, and solvent-to-material ratio. Table 2shows the effect of temperature on TPC and antioxidant activity.The highest phenolics content and AA were measured in the ex-tracts at 75 ◦C with a trend for greater extraction of phenolics andantioxidants with increased temperature. UAE extracts at 40 ◦Chad TPC of 8.85 ± 0.33 mg SAE/g meal, which was equal toconventional hot ethanolic extracts (Table 1) at 8.81 ± 0.17 mgSAE/g meal (P > 0.05). UAE extracts at 75 ◦C had significantlygreater TPCs and ABTS antioxidant activities than conventionalhot-solvent extracts (Table 1) (P < 0.05).

The effect of sonication duration on TPC and antioxidant ac-tivity is presented in Table 3. Sonication for longer than 30 mindid not increase the concentration of phenolics in extracts. Twentyminutes of sonication were sufficient to maximize antioxidant ex-traction as determined in both the DPPH and ABTS assays. Table 4indicates that water alone was sufficient in extracting phenolicsand compounds with ABTS antioxidant activity, and no advan-tage was gained by adding ethanol. However, inclusion of ethanolimproved the extraction of compounds with DPPH antioxidant

Table 4–Effect of extraction solvent EtOH concentration on TPCand antioxidant activity as measured in UAE extracts from B.juncea seed meal (20 ◦C, 60 W, 120 mL, 50 : 1 solvent, 30 min).a

Antioxidant activity(mg SAE/g meal)

EtOH Total phenolics(%) (mg SAE/g meal) DPPH assay ABTS assay

0 8.38 ± 0.12a 4.84 ± 0.06b 5.62 ± 0.13a30 7.41 ± 0.23b 5.12 ± 0.59b 5.62 ± 0.18a50 7.55 ± 0.11b 5.45 ± 0.93b 5.61 ± 0.25a70 8.38 ± 0.28a 6.57 ± 0.24a 5.47 ± 0.07a95 2.62 ± 0.32c 1.86 ± 0.47c 1.67 ± 0.26b


Table 5–Effect of extraction solvent-to-material ratio on TPCand antioxidant activity as measured in UAE extracts from B.juncea seed meal (20 ◦C, 70% EtOH, 60 W, 120 mL, 30 min).a

Antioxidant activitySolvent-to- Total phenolics (mg SAE/g meal)material ratio (mg SAE/g(mL/g) meal) DPPH assay ABTS assay

10 6.38 ± 0.20c 4.41 ± 0.23c 3.85 ± 0.14c20 6.90 ± 0.21c 4.75 ± 0.17c 5.09 ± 0.17b30 7.60 ± 0.40ab 5.69 ± 0.68b 5.43 ± 0.26ab40 7.93 ± 0.48ab 6.43 ± 0.18a 5.42 ± 0.24ab50 8.38 ± 0.28a 6.57 ± 0.24a 5.47 ± 0.07ab60 8.14 ± 0.19ab 6.57 ± 0.18a 5.63 ± 0.37a

aValues are means of at least 3 measurements ± standard deviation with meal moisturecontent between 4% and 5% (wet basis). Means within a column followed by the sameletter are not significantly different (P < 0.05).

activity, with maximum extraction of such compounds occurringwhen 70% ethanol was used. The effect of solvent-to-materialratio on TPC and AA is presented in Table 5. There was a trendtoward greater extraction of phenolics and antioxidants at highersolvent ratios and a ratio of 40 : 1 was sufficient to maximize theextraction of all 3 components. The most significant UAE param-eters included the solvent-to-material ratio, solvent composition,and extraction temperature.

Correlations among antioxidant assaysSignificant linear correlation (P < 0.05) was detected in all cases

for the TPC, DPPH, and ABTS antioxidant assays. The ABTS as-say correlated more closely with the TPC (r = 0.912) than with theDPPH assay (r = 0.603), and the TPC and DPPH assay were leastcorrelated (r = 0.494). Some studies have reported excellent linearcorrelations between antioxidant activity assays and TPC (Huangand others 2005; Hassas-Roudsari and others 2009), while oth-ers have reported a lack of correlation (Costa and others 2009).Wanasundara and others (1995) analyzed canola meal and con-cluded that the TPC was not the critical factor in determining theantioxidant activity. Costa and others (2009) reported that the TPCmay be an indicator of potential antioxidant activity, but there isnot necessarily a linear correlation. In our study, the correlationsare heavily influenced by the suspected difference in composi-tion between the alcoholic and aqueous extracts, thus raising thequestion of whether to use alcohol in the extraction solvent. Wa-ter would be preferred from an environmental perspective, butdrawbacks could include greater extraction of undesirable com-pounds such as proteins, more extensive filtration requirements ofthe extraction solution, and lower yield of specific antioxidants.




Table 6–Extract yields (g/g meal) obtained from different extrac-tion methods as measured in extracts from B. juncea seed meal.a

Extract Extract yield (g extract/g meal)

Water 7 db 0.33 ± 0.01aWater 24 hb 0.29 ± 0.01b70% EtOH 7 dc 0.23 ± 0.01cHot 70% EtOHd 0.22 ± 0.01cd75 ◦C UAEe 0.22 ± 0.01cd70% EtOH 24 hc 0.21 ± 0.01dHot waterf 0.21 ± 0.01d20 ◦C UAEe 0.18 ± 0.01e

aValues are means of at least 3 measurements ± standard deviation. Meal moisturecontent was between 4% and 5% (wet basis). Means within a column followed by thesame letter are not significantly different (P < 0.05).bLong-duration water extraction at 25 ◦C for either 24 h or 7 d.cLong-duration 70% EtOH (v/v) extraction at 25 ◦C for either 24 h or 7 d.dHot 70% EtOH (v/v) extraction at 80 ◦C for 3×30 min.eUAE at 20 kHz, 20 W/cm2, 70% EtOH (v/v), and at either 20 ◦C or 75 ◦C for 30 min.fHot water extraction at 80 ◦C for 3×30 min.

Extract yieldsThe extract yields, on a dried mass basis, from conventional and

sonicated extraction methods are presented in Table 6. The yieldsranged from 0.18 ± 0.01 to 0.33 ± 0.01 g/g meal. The lowest yieldwas obtained from UAE extracts at 20 ◦C and the highest yieldwas obtained from 7-d aqueous extracts. UAE extracts at 75 ◦Chad the same yield as those of conventional ethanolic extracts, buthad higher TPC and antioxidant activity. The long-duration aque-ous extracts had the highest yields, possibly due to the increasedsolubility of protein in water compared to ethanol. It appears thatUAE with ethanolic solvents is an effective technique to increasethe extraction of antioxidants without significantly increasing themass of the extracts.

Comparison among BSM extractsThe TPC and antioxidant activity of various BSM extracts are

presented in Table 7. The sinapine contents of BSM vary widely,

Table 7–Results of conventional extraction of various BSM.a

Total phenolics DPPH assay ABTS assay(mg SAE/g (mg SAE/g (mg SAE/g

Seed meal meal) meal) meal)

B. juncea 8.81 ± 0.17 7.34 ± 0.37 5.98 ± 0.22S. alba 39.11 ± 0.38 8.70 ± 0.16 8.33 ± 0.16B. napus 14.53 ± 0.17 8.25 ± 0.16 9.19 ± 0.14

aHot 70% EtOH (v/v) extraction at 80 ◦C for 3×30 min.

with measured values in batches of B. juncea and B. napus mealsranging from 7.3 to 14.3 mg/g and 7.4 to 16.2 mg/g, respectively(Emery and others 2003).

It is noteworthy that S. alba seemingly has considerably higherTPC than either B. juncea or B. napus, but does not appear tohave correspondingly high AA (Table 7). S. alba meal contains4-hydroxybenzyl glucosinolate, which has been measured at levelsgreater than 120 µmol/g meal (Borek and Morra 2005). Basedon calculation of molar equivalence, 120 µmol 4-hydroxybenzylglucosinolate would have the same phenolic content as 27 mgsinapic acid.

Sinapine and sinapic acidThe chromatograms of aqueous extracts (Figure 1) indicate that

major components of the extracts are sinapine (SP) and sinapicacid (SA). Hot water extraction resulted in extracts with a highconcentration of sinapine with minimal sinapic acid. However,if the extract was allowed to remain in water at 20 ◦C for 7 d,the concentration of sinapine diminished while that of sinapicacid increased greatly. Sinapine is susceptible to both alkaline andenzymatic (myrosinase) hydrolysis (Durkee and Thivierge 1975),producing sinapic acid and choline, and this may explain thesechanges in extract composition.

The results of the extraction kinetics of sinapine during sonica-tion are presented in Figure 2. The extraction of sinapine during

Figure 1–Comparison of the HPLC chromatograms ofhot water extraction (a) and 7-d aqueous extractionat 20 ◦C (b). SP and SA are sinapine and sinapic acidpeaks, respectively.


E:Fo

odEn

ginee

ring&

Phys

icalP

ropert

ies


sonication at 75 ◦C was greater than that at 20 ◦C (P < 0.05).The sinapine content positively correlated with the TPCs andantioxidant activities of the UAE extracts with increasing tem-perature, suggesting that sinapine may be an important pheno-lic antioxidant found in B. juncea meal ethanolic extracts. Otherstudies have reported a similar correlation between sinapine andTPC in BSM (Khattab and others 2010; Thiyam and others2006).

Figure 3 shows the 1st-order degradation kinetics for the 4sinapine and sinapic acid solutions. The corresponding rate con-stants for the compounds are: pure sinapic acid (k = 0.001662) >

pure sinapine (k = 0.001045) > sinapic acid in extract (k =0.0008542) > sinapine in extract (k = 0.0001278). Sinapine inextracts showed negligible degradation during sonication (P >

0.05), whereas all other situations resulted in significant degra-dation (P < 0.05). Sinapic acid appears to be more susceptibleto sonochemical degradation than sinapine. Less degradation ofboth sinapic acid and sinapine occurred when present in extractscontaining various unidentified components as compared to thesame compound when present as the sole component of the soni-cated solution. These results suggest that the susceptibility of thesecompounds to the degradation effects of ultrasonic cavitation maydepend on their molecular composition and whether they arepresent with other components in solution.

0

2

4

6

8

10

0 10 20 30 40 50 60

Sina

pine

(mg

/ g

Mea

l)

Sonica�on Dura�on (min)

Figure 2–HPLC analysis of the extraction kinetics of sinapine during UAEat 20 ◦C (�) and 75 (•). The extraction of sinapine is expressed as mass ofsinapine in extract per mass meal extracted.

0.88

0.90

0.92

0.94

0.96

0.98

1.00

0 10 20 30 40 50 60

Ct/C

o

Sonica�on Dura�on (min)

Figure 3–Degradation kinetics of sinapine and sinapic acid during sonica-tion at 20 ◦C: pure sinapine (�), sinapine in extract (X), pure sinapic acid(�), and sinapic acid in extract (•).

ConclusionThe data from this study suggest that ultrasound treatment can

assist the extraction of antioxidants from B. juncea seed meal byreducing both the temperature and duration requirement withoutsignificant degradation of one of the primary antioxidants present.Future work will be focused on incorporating the antioxidant-richBSM extracts in a meat product to inhibit spoilage and prolongshelf life. The use of mustard meal for this purpose is especiallyappealing because these meals also contain glucosinolates that hy-drolyze to form biologically active compounds. For example, 2-propenyl glucosinolate in B. juncea seed meal is enzymatically hy-drolyzed to a volatile 2-propenyl isothiocyanate with antimicrobialproperties (Lin and others 2000; Lara-Lledo and others 2012). Ifantioxidants and antimicrobials can be obtained in a single ex-tract from mustard seed meal, then both chemical and microbialspoilage of foods can be inhibited.

AcknowledgmentsThis project was supported by USDA Natl. Inst. of Food and

Agriculture competitive grant nr 2011-67009-20094. We thankDr. Greg Moller (Univ. of Idaho, School of Food Science) forproviding the high-frequency ultrasound equipment used in thestudy.

ReferencesAlbu S, Joyce E, Paniwnyk L, Lorimer JP, Mason TJ. 2004. Potential for the use of ultrasound

in the extraction of antioxidants from Rosmarinus officinalis for the food and pharmaceuticalindustry. Ultrason Sonochem 11(3-4):261–65.

Amarowicz R, Pegg RB, Rahimi-Moghaddam P, Barl B, Weil JA. 2004. Free-radical scavengingcapacity and antioxidant activity of selected plant species from the Canadian prairies. FoodChem 84(4):551–62.

Borek V, Morra MJ. 2005. Ionic thiocyanate (SCN-) production from 4-hydroxybenzyl glu-cosinolate contained in Sinapis alba seed meal. J Agric Food Chem 53(22):8650–4.

Bouchereau A, Hamelin J, Lamour I, Renard M, Larher F. 1991. Distribution of sinapine andrelated-compounds in seeds of brassica and allied genera. Phytochemistry 30(6):1873–81.

Branen AL. 1975. Toxicology and biochemistry of butylated hydroxyanisole and butylated hy-droxytoluene. J Am Oil Chemists’ Soc 52(2):59–63.

Brettonnet A, Hewavitarana A, DeJong S, Lanari MC. 2010. Phenolic acids composition and an-tioxidant activity of canola extracts in cooked beef, chicken and pork. Food Chem 121(4):927–33.

Brown J, Davis JB, Brown DA, Seip L, Gosselin T. 2004. Registration of ‘Pacific Gold’ orientalcondiment mustard. Crop Sci 44(6):2271–2.

Brown J, Davis JB, Erickson DA, Brown AP, Seip L. 1998. Registration of ‘IdaGold’ yellowmustard. Crop Sci 38(2):541.

Contamine RF, Wilhelm AM, Berlan J, Delmas H. 1995. Power measurement in sonochemistry.Ultrason Sonochem 2(1):S43–7.

Costa RM, Magalhaes AS, Pereira JA, Andrade PB, Valentao P, Carvalho M, Silva BM. 2009.Evaluation of free radical-scavenging and antihemolytic activities of quince (Cydonia oblonga)leaf: a comparative study with green tea (Camellia sinensis). Food Chem Toxicol 47(4):860–5.

Durkee AB, Thivierge PA. 1975. Bound phenolic acids in brassica and sinapis oilseeds. J FoodSci 40(4):820–2.

Emery RJ, Papadaki M, Mantzavinos D. 2003. Sonochemical degradation of phenolic pollutantsin aqueous solutions. Environ Technol 24(12):1491–500.

Engels C, Schieber A, Ganzle MG. 2012. Sinapic acid derivatives in defatted Oriental mustard(Brassica juncea L.) seed meal extracts using UHPLC-DAD-ESI-MSn and identification ofcompounds with antibacterial activity. Eur Food Res Technol 234(3):535–42.

Esclapez MD, Garcia-Perez JV, Mulet A, Carcel JA. 2011. Ultrasound-assisted extraction ofnatural products. Food Eng Rev 3(2):108–20.

Hassas-Roudsari M, Chang PR, Pegg RB, Tyler RT. 2009. Antioxidant capacity of bioactivesextracted from canola meal by subcritical water, ethanolic and hot water extraction. FoodChem 114(2):717–26.

Hendrix KM, Morra MJ, Lee HB, Min SC. 2012. Defatted mustard seed meal-based biopolymerfilm development. Food Hydrocolloids 26(1):118–25.

Hoffmann MR, Hua I, Hochemer R. 1996. Application of ultrasonic irradiation for the degra-dation of chemical contaminants in water. Ultrason Sonochem 3(3):163–72.

Huang DJ, Ou BX, Prior RL. 2005. The chemistry behind antioxidant capacity assays. J AgricFood Chem 53(6):1841–56.

Kentish S, Ashokkumar M. 2011. The physical and chemical effects of ultrasound. In: Feng H,Barbosa-Canovas GV, Weiss J, editors. Ultrasound technologies for food and bioprocessing.New York: Springer. p 1–12.

Khattab R, Eskin M, Aliani M, Thiyam U. 2010. Determination of sinapic acid derivativesin canola extracts using high-performance liquid chromatography. J Am Oil Chemists’ Soc87(2):147–55.

Knorr D, Zenker M, Heinz V, Lee DU. 2004. Applications and ultrasonics in food potential ofprocessing. Trends Food Sci Technol 15(5):261–6.

Kozlowska H, Rotkiewicz DA, Zadernowski R. 1983. Phenolic-acids in rapeseed and mustard.J Am Oil Chemists’ Soc 60(6):1119–23.




Lara-Lledo M, Olaimat A, Holley RA. 2012. Inhibition of Listeria monocytogenes on bolognasausages by an antimicrobial film containing mustard extract or sinigrin. Intl J Food Microbiol156(1):25–31.

Lin CM, Preston JF, Wei CI. 2000. Antibacterial mechanism of allyl isothiocyanate. J Food Prot63(6):727–34.

Mailer RJ, McFadden A, Ayton J, Redden B. 2008. Anti-nutritional components, fibre, sinapineand glucosinolate content, in Australian canola (Brassica napus L.) meal. J Am Oil Chemists’Soc 85(10):937–44.

Mason TJ, Paniwnyk L, Lorimer JP. 1996. The uses of ultrasound in food technology. UltrasonSonochem 3(3):S253–60.

Naczk M, Pegg RB, Zadernowski R, Shahidi F. 2005. Radical scavenging activity of canolahull phenolics. J Am Oil Chemists’ Soc 82(4):255–60.

Peterson CL, Auld DL, Thompson JC. 1983. Experiments with vegetable oil expression. TransASAE 26(5):1298–302.

Pokorny J. 2007. Are natural antioxidants better and safer than synthetic antioxidants? Eur J LipidSci Technol 109(6):629–42.

Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. 1999. Antioxidantactivity applying an improved ABTS radical cation decolorization assay. Free Radical BiolMed 26(9–10):1231–7.

Rice-Evans CA, Miller NJ, Paganga G. 1996. Structure-antioxidant activity relationships offlavonoids and phenolic acids. Free Radical Biol Med 20(7):933–56.

Saleemi ZO, Janitha PK, Wanasundara PD, Shahidi F. 1993. Effects of low-pungency groundmustard seed on oxidative stability, cooking yield, and color characteristics of comminutedpork. J Agric Food Chem 41(4):641–3.

Shahidi F, Wanasundara UN, Amarowicz R. 1994. Natural antioxidants from low-pungencymustard flour. Food Res Intl 27(5):489–93.

Soria AC, Villamiel M. 2010. Effect of ultrasound on the technological properties and bioactivityof food: a review. Trends Food Sci Technol 21(7):323–31.

Thiyam U, Stockmann H, Felde TZ, Schwarz K. 2006. Antioxidative effect of the mainsinapic acid derivatives from rapeseed and mustard oil by-products. Eur J Lipid Sci Tech-nol 108(3):239–48.

Thompson LH, Doraiswamy LK. 1999. Sonochemistry: science and engineering. Ind Eng ChemRes 38:1215–49.

Vilkhu K, Mawson R, Simons L, Bates D. 2008. Applications and opportunities for ultrasoundassisted extraction in the food industry – a review. Innov Food Sci Emerg Technol 9(2):161–9.

Vinatoru M. 2001. An overview of the ultrasonically assisted extraction of bioactive principlesfrom herbs. Ultrason Sonochem 8(3):303–13.

Wanasundara PKJPD, Shahidi F. 2005. Antioxidants: science, technology and applications. In:Shahidi F, editor. Bailey’s industrial oil and fat products. 6th ed. New York: John Wiley &Sons. p 431–89.

Wanasundara UN, Amarowicz R, Shahidi F. 1995. Partial characterization of natural antioxidantsin canola meal. Food Res Intl 28(6):525–30.

Wanasundara UN, Shahidi F. 1994. Canola extract as an alternative natural antioxidant for canolaoil. J Am Oil Chemists’ Soc 71(8):817–22.

Wang T, Liang H, Yuan Q. 2011. Optimization of ultrasonic-stimulated solvent extraction ofSinigrin from Indian mustard seed (Brassica juncea L.) using response surface methodology.Phytochem Anal 22(3):205–13.


Antioxidant Extraction from Mustard ( Brassica juncea ) Seed Meal Using...

Documents

Transcript of Antioxidant Extraction from Mustard ( Brassica juncea ) Seed Meal Using...