Food Chemistry 126 (2011) 1122–1126

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Food Chemistry

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Antioxidant activities and total phenolic contents of various extracts fromdefatted wheat germ

Ke-Xue Zhu ⇑, Cai-Xia Lian, Xiao-Na Guo, Wei Peng, Hui-Ming ZhouState Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi-214122, Jiangsu Province, PR China

a r t i c l e i n f o

Article history:Received 20 June 2010Received in revised form 11 November 2010Accepted 24 November 2010Available online 2 December 2010

Keywords:Antioxidant activityFree radical scavengingDefatted wheat germ extractsTotal phenolic contents

0308-8146/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.foodchem.2010.11.144

⇑ Corresponding author. Tel./fax: +86 510 8532903E-mail address: kxzhu@jiangnan.edu.cn (K.-X. Zhu

a b s t r a c t

Defatted wheat germ (DWG) is the main by-product of the wheat germ oil extraction process. Its nutri-tional value has well been accepted. In this study, the antioxidant properties of 30% ethanol, 50% ethanol,70% ethanol, 100% ethanol, and aqueous extracts of DWG were measured using various in vitro assays.Among the DWGEs (DWG extracts) tested, the 70% ethanol extract showed the best DPPH radical scav-enging power while the 100% ethanol extract showed the highest ABTS radical scavenging activity andreducing power. In addition, both the 70% ethanol extract and the 50% ethanol extract exhibited relativelyhigher antioxidant activity in linoleic acid system. The extracts in question exhibited total phenolic con-tents ranging from 13.98 to 16.75 mg GAE/g. DWG, as a source of natural antioxidants, can be used toformulate nutraceuticals with potential applications to reducing the level of oxidative stress. The antiox-idant potency of the DWG extracts could be the basis for its health promoting potential.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Natural antioxidants, which are ubiquitous in fruits, teas, vege-tables, cereals, and medicinal plants, have received great attentionand have been studied extensively, since they are effective freeradical scavengers and are assumed to be less toxic than syntheticantioxidants, such as butylated hydroxyanisole (BHA) and butyl-ated hydroxyltoluene (BHT), which are suspected of being carcino-genic and causing liver damage (Ratnam, Ankola, Bhardwaj,Sahana, & Kumar, 2006). It is believed that an increased intake offood, which is rich in natural antioxidants is associated with a low-er risk of degenerative diseases, particularly cardiovascular dis-eases and cancer (Perez-Jimenez et al., 2008).

Wheat is one of the major cereals and food ingredients acrossthe world. Wheat kernel is composed of bran, germ, and endo-sperm morphologically. The antioxidants in wheat include carote-noids, tocopherols, flavonoids and phenolic acids. In the past years,the antioxidant activities of whole wheat and milling fractions(Liyana-Pathirana & Shahidi, 2007; Vaher, Matso, Levandi, Helmja,& Kaljurand, 2010; Zhou, Su, & Yu, 2004) have been widely studied.Antioxidant rich extracts have been obtained from wheat usingvarious solvents including water, ethanol, methanol, an aqueousethanol solution, and an aqueous methanol solution (Vaher et al.,2010; Zielinski, 2000). Some studies have also reported on the anti-oxidant activity of roasted defatted wheat germ (Gelmez, Kıncal, &Yener, 2009; Krings, El-Saharty, El-Zeany, Pabel, & Berger, 2000),

ll rights reserved.

7.).

wheat germ oils (Malecka, 2002), and wheat germ protein hydrol-ysates (Zhu, Zhou, & Qian, 2006b).

Defatted wheat germ (DWG), the main by-product in the wheatgerm oil extraction process, contains many nutritional ingredients,such as proteins, carbohydrates, B vitamins, pigments, and miner-als and some functional microcomponents (Ge, Sun, Ni, & Cai,2000; Zhu, Zhou, & Qian, 2006a). However, few systematic studieshave been found on WG antioxidants. Furthermore, the phenolicsin WG have been less treated in terms of their contents and contri-bution to the overall antioxidant activities of WG. As a matter offact, due to the complex nature of phytochemicals, their antioxi-dant activity should be evaluated using several commonlyaccepted assays.

In the present study, defatted wheat germ extracts (DWGEs)were obtained using water, aqueous ethanol solutions and ethanolas solvents, and each determined for its antioxidant activities byin vitro methods, including DPPH scavenging assay, ABTS scaveng-ing assay, reducing power, and antioxidant activity in a linoleicacid system, and for its total phenolic contents by the method ofFolin–Ciocalteu reagent.

2. Materials and methods

2.1. Materials and chemicals

Raw wheat germ (RWG) was a gift from Huai’an Xinfeng FlourMill (Jiangsu, China). 2,20-azinobis-3-ethylbenzothiazoline-6-sul-phonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)were purchased from Sigma Chemical Co. (St. Louis, USA). Linoleic

K.-X. Zhu et al. / Food Chemistry 126 (2011) 1122–1126 1123

acid was from Zhongchuang Bio-engineering Co., Limited. Otherchemicals were of analytical grade.

2.2. Preparation of defatted wheat germ flour (DWGF)

After being removed of contaminants, RWG was defatted withn-hexane until the supernatant was colourless, and then the result-ing DWG was consecutively air-dried at room temperature, re-moved of the detached fine debris by sieving through a 60-meshscreen, and milled by a laboratory hammer mill to a fineness of100-mesh. The DWGF was kept in sealed bags at 4 �C until use.

2.3. Preparation of the extracts

Aliquots of DWGF 5 g each was extracted with the followingsolvents: water, ethanol/water (30%, by volume), ethanol/water(50%, by volume), and ethanol/water (70%, by volume), and etha-nol. The extraction involved two steps of extraction under gentlestirring at room temperature, respectively, using 50 ml solventfor 3 h and 25 ml solvent for 1 h, each followed by centrifugationat 10,000 rpm, 4 �C for 20 min, to obtain the supernatant. Thesupernatant derived from both steps was combined, concentratedunder vacuum at 50 �C, and freeze-dried. The resultant powderwas stored in sealed containers at 4 �C for further analysis.Theextraction yield was calculated by the following equation:

Extraction yield%

¼ f½weightof freeze� dried powderðgÞ�=½weight of DWGFðgÞ�g � 100

2.4. DPPH radical scavenging assay

The DPPH radical scavenging assay was conducted according tothe method of Zhu et al. (2006b). Briefly, 2 ml of DPPH solution(0.1 mM, in ethanol) was mixed with 2 ml of the samples dissolvedin the extracting solvent at different concentrations (0.4–1.2 mg/ml). The reaction mixture was shaken and incubated in the darkat room temperature for 60 min, and the absorbance was read at517 nm against the blank. Controls were prepared in a similarway as for the test group except for the replacement of the antiox-idant solution with the corresponding extraction solvent. The inhi-bition of the DPPH radical by the sample was calculated accordingto the following formula:

DPPHscavengingactivityð%Þ

¼ Abs: of control� Abs: of sampleAbs: of control

� 100%

2.5. ABTS radical scavenging assay

The ABTS radical scavenging assay was done according to themethod of Re et al. (1999) with slight modifications. The ABTS rad-ical was generated by the oxidation of ABTS with potassium per-sulphate. The ABTS radical cation solution was obtained asfollows: Five millilitre of ABTS (7 mM) was mixed with 88 ll ofpotassium persulphate (140 mM) and then incubated in the darkat room temperature for 12–16 h. The working solution was pre-pared by diluting the previous solution with phosphate bufferedsaline (100 ml pH 7.4 PBS containing 81.0 ml Na2HPO4 (0.2 M)and 19.0 ml NaH2PO4 (0.2 M) until the absorbance at 734 nm was0.70 ± 0.02. The solution was kept for 30 min in the dark beforebeing used. One hundred and fifty micro liters of each samplewas mixed with 2.85 ml of the working solution, shaken vigor-ously, and left to stand for 10 min at room temperature. The absor-bance of the reaction mixture was determined at 734 nm. The

controls contained the extraction solvent instead of the antioxi-dant solution. The ABTS radical scavenging capacity of the samplewas calculated by the following formula:

ABTS radical scavenging activity ð%Þ

¼ Abs: ofcontrol� Abs: ofsampleAbs: ofcontrol

� 100%

2.6. Reducing power

The reducing power of the samples was determined accordingto the method of Atmani et al. (2009). One milliliter of each samplewas mixed with 2.0 ml of phosphate buffered saline (0.2 M, pH 6.6)and 2.5 ml of potassium ferrocyanate (1%, w/v). The mixture wasincubated at 50 �C for 20 min. Then, 2.5 ml of trichloroacetic acid(10%) was added to the mixture. A portion of the solution(2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride(0.5 ml, 0.1%), and the absorbance was measured at 700 nm at areaction time of 30 min.

2.7. Antioxidant activity in a linoleic acid system

The inhibition on linoleic acid peroxidation was assayed accord-ing to the ferric thiocyanate (FTC) method with some modifications(Liu & Yao, 2007). The sample (200 lg) in water (2 ml) was addedto a solution mixture of 2.5% linoleic acid in ethanol (2 ml), phos-phate buffer (4 ml, 0.05 M, pH 7.0) and distilled water (2 ml). Themixture was incubated in darkness at 40 �C. fifty micro liters ofthe incubation solution was mixed with 75% ethanol (4 ml), 30%ammonium thiocyanate (50 ll) and 20 mM ferrous chloride in3.5% hydrochloric acid (50 ll). After 3 min at room temperature,the absorbance of the mixture was determined at 500 nm. The con-trol contained water instead of the sample. The per cent inhibitionon linoleic acid peroxidation was calculated as:

Inhibitionð%Þ ¼ Abs: of control� Abs: of sampleAbs: of control

� 100%

2.8. Total phenolic content

The total phenolic content was determined by the method ofFolin–Ciocalteu reagent (FCR). The Folin–Ciocalteu reagent wasfreshly prepared according to the method of Zhou et al. (2004)by refluxing a mixture of 12.5 g sodium molybdate, 50 g sodiumtungstate, 25 ml phosphoric acid (85%), 50 ml concentrated hydro-chloric acid and 350 ml water for 10 h followed successively byreacting it with 75 g lithium sulphate and oxidising it by a fewdrops of bromine. The total volume of the resulting solution wasadjusted to 500 ml with distilled water. The solution was filteredand stored at 4 �C in the dark.

Half a milliliter of each sample (1 mg/ml) was mixed with2.5 ml of diluted (1:10 with water) Folin–Ciocalteu reagent and2 ml of sodium carbonate solution (7.5%, w/v). After 90 min ofincubation at 30 �C, the absorbance was measured at 765 nm.The total phenolic content of the extract was calculated by com-parison with a standard curve generated by analysing gallic acid(Bahramikia, Ardestani, & Yazdanparast, 2009).

2.9. Statistical analyses

The data obtained in this study were expressed as the mean ofthree replicate determinations plus or minus the standard devia-tion (SD). Statistical comparisons were made with Student’s test.P values <0.05 were considered to be significant.

1124 K.-X. Zhu et al. / Food Chemistry 126 (2011) 1122–1126

3. Results and discussion

3.1. Extraction yields and total phenolic contents of extracts

Extraction is an important step for obtaining extracts withacceptable yields and strong antioxidant activity (Moure et al.,2001). In this experiment, the yields of DWGE ranged from 5.16%for the ethanol extract to 48.03% for the aqueous extract (Table1), depending on the extraction solvent in the following order:water > 30% ethanol > 50% ethanol > 70% ethanol > 100% ethanol.The yield of 50% ethanol extract did not show significant differencewith the 30% and 70% ethanol extracts; but the yield of the aque-ous extract was significantly (P < 0.05) different from the 100% eth-anol extract, which might be attributable to the higher solubility ofproteins and carbohydrates in water than in ethanol and ethanol/water mixtures, as found by Zielinski and Kozłowska (2000).

The total phenolic contents of the extracts, as obtained from thecalibration curve (y = 74.945x � 4.5821, R2 = 0.9993, x is the absor-bance; y is the concentration of gallic acid solution, lg/ml) and ex-pressed as gallic acid equivalents (mg gallic acid/g dried extract),ranged from 13.98 mg GAE/g for the 30% ethanol extract to16.75 mg GAE/g for the 70% ethanol extract (Table 1); 70% ethanolwas regarded as the most effective solvent for extracting phenolicsfrom DWGF. The total phenolic contents of the aqueous extractwere higher than those of the 30% ethanol extract and the ethanolextract. This could be explained by the possible formation of com-plexes by a part of the phenolic compounds with carbohydratesand proteins, which are more extractable in water than in 30% eth-anol or ethanol (Bonoli, Marconi, & Caboni, 2004; Moure et al.,2001; Zhao & Hall, 2008).

3.2. DPPH radical scavenging activity

DPPH radical is a stable organic free radical with an absorptionband at 517 nm. It loses this absorption on accepting an electron ora free radical species, which results in a visually noticeable discol-ouration from purple to yellow. It can accommodate many samplesin a short period and is sensitive enough to detect active ingredi-ents at low concentrations (Hseu et al., 2008). In this study, allthe extracts showed DPPH scavenging activities in a concentra-

Table 1The yields and total phenolics contents of DWGEs.

Sample Extraction %(w/w)

Total phenolics content(mg GAE/g)

Aqueous extract 48.03 ± 1.33d 14.63 ± 0.04ab30% ethanol extract 33.88 ± 0.17c 13.98 ± 0.31a50% ethanol extract 32.39 ± 0.26bc 15.95 ± 0.27c70% ethanol extract 31.87 ± 0.03b 16.75 ± 0.28d100% ethanol extract 5.16 ± 0.02a 14.93 ± 0.30b

Values are shown as mean ± standard deviation of three replicates. Means followedby the same letter in the same column are not significantly different (P < 0.05).

0102030405060708090

0.4 0.6 0.8 1 1.2Concentration (mg/ml)

Scav

engi

ng e

ffec

t on

DPP

Hra

dica

ls (

%) Aqueous extract

30% Ethanol extract50% Ethanol extract70% Ethanol extract100% Ethanol extract

Fig. 1. DPPH radical scavenging activities of DWGEs.

tion-dependent manner whose profiles varied among the differentextracts (Fig. 1). The 70% ethanol extract showed the highest DPPHradical scavenging activity at all concentrations, while the 30% eth-anol extract demonstrated acutely increasing DPPH radical scav-enging activity. The ethanol extract had stronger scavengingcapacity than the aqueous extract. A similar trend was observedin a study of the antioxidant activity of the field horsetail (tsukushi)Equisetum arvense L. extracts (Nagai, Myoda, & Nagashima, 2005).

3.3. ABTS radical scavenging activity

The ABTS radical cation decolourisation test is another methodwidely used to assess antioxidant activity. Reduction in colour indi-cates reduction of ABTS radical (Adedapo, Jimoh, Koduru, Masika, &Afolayan, 2008). As shown in Fig. 2, all the extracts reduced theabsorbance at 734 nm, and the concentration of the extracts was di-rectly proportional to the reduction. The aqueous extract showedthe lowest ABTS radical scavenging activity (Maisuthisakul,Pongsawatmanit, & Gordon, 2007). The extracts, at the low concen-trations of 5.0 and 7.5 mg/ml, had ABTS radical scavengingactivities in the order: 100% ethanol extract > 30% ethanolextract > 50% ethanol extract > 70% ethanol extract > aqueousextract. But at other concentrations, the difference among the activ-ities of the extracts was indistinctive except for the aqueous extract.

3.4. IC50 of DPPH and ABTS

The IC50 value was defined as the concentration of the samplenecessary to cause 50% inhibition, which was obtained by interpo-lation from linear regression analysis (Yang et al., 2010). A lowerIC50 value is associated with a higher radical scavenging activity.It was found that 70% ethanol extract had the strongest DPPH rad-ical scavenging activity, while 100% ethanol extract showed thestrongest ABTS radical scavenging activity (Table 2). There weresignificant differences (P < 0.05) in the DPPH radical scavengingactivity among the extracts except the aqueous extract and 30%ethanol extract. In the ABTS radical scavenging assay, significantdifferences among the IC50 values of all the extracts were found(P < 0.05).

0

20

40

60

80

100

5 7.5 10 12.5 15concentration (mg/ml)

Scav

engi

ng e

ffec

t on

AB

TS

radi

cal (

%)

Aqueous extract

30% Ethanol extract

50% Ethanol extract

70% Ethanol extract

100% Ethanol extract

Fig. 2. ABTS radical scavenging activities of DWGEs.

Table 2IC50 in radical scavenging activities of DWGE.

Samples IC50 of DPPH radical(mg/ml)

IC50 of ABTS radical(mg/ml)

Aqueous extract 0.814 ± 0.006d 9.37 ± 0.05e30% ethanol extract 0.797 ± 0.007d 6.71 ± 0.01b50% ethanol extract 0.703 ± 0.006c 7.03 ± 0.09c70% ethanol extract 0.424 ± 0.006a 7.84 ± 0.04d100% ethanol extract 0.593 ± 0.006b 5.6 ± 0.06a

Values are shown as mean ± standard deviation of three replicates. Means followedby the same letter in the same column are not significantly different (P < 0.05).

0.00.20.40.60.81.01.21.41.61.8

0 1 2 3 4 5 6 7 8

Incubation time(day)

Abs

orba

nce

at 5

00 n

m

Control

Aqueous extract

30% Ethanol extract

50% Ethanol extract

70% Ethanol extract

100% Ethanol extract

Fig. 4. Antioxidant activity of DWGEs in linoleic acid system.

K.-X. Zhu et al. / Food Chemistry 126 (2011) 1122–1126 1125

The order of IC50 of DPPH radical was inconsistent with that ofthe ABTS radical. It should be mentioned that DPPH and ABTS as-says were carried out in ethanol and aqueous media, respectively.ABTS radical scavenging is less susceptible to steric hindrance(Delgado-Andrade, Rufian-Henares, & Morales, 2005; Mai-suthisakul et al., 2007). From a mechanistic standpoint, the DPPHradical scavenging assay could reflect the capacity of the extracttransferring electrons or hydrogen atoms, while the ABTS radicalscavenging activity could reflect the hydrogen donating and thechain-breaking capacity of the extract (Perez-Jimenez et al., 2008).

3.5. Reducing power

The reducing power of the extracts, which may serve as asignificant reflection of antioxidant activity, was determined usinga modified Fe3+ to Fe2+ reduction assay, whereby the yellow colourof the test solution changes to various shades of green and blue,depending on the reducing power of the samples. Thepresence of antioxidants in the samples causes the reduction ofthe Fe3+/ferricyanide complex to the Fe2+ form, and Fe2+ can bemonitored by measurement of the formation of Perl’s Prussian blueat 700 nm (Yang et al., 2010). In Fig. 3, all the extracts showedsome degree of electron-donating capacity in a linear concentra-tion-dependent manner. The reducing power followed the order:100% ethanol extract > 70% ethanol extract > 50% ethanol ex-tract > 30% ethanol extract > aqueous extract. The reducing powerof the ethanol extract was superior to that of the aqueous extract,which coincides with other reports (Kumar, Ganesan, & Rao, 2008;Yildirim, Mavi, & Kara, 2001).

3.6. Antioxidant activity in a linoleic acid system

In the present study, the antioxidant activity of DWGEs wasdetermined by its retarding effects on linoleic acid peroxidationusing the thiocyanate method. Peroxides resulting from linoleicacid peroxidation can oxidise Fe2+ to Fe3+; Fe3+ forms a complexwith SCN�, which has a maximum absorbance at 500 nm. Highabsorbance is an indication of high concentration of peroxideformed during the emulsion incubation (Liu & Yao, 2007; Atmaniet al., 2009). The antioxidant activities of the extracts in the linoleicacid system are shown in Fig. 4. Compared with the control, all theextracts could inhibit linoleic acid peroxidation. The inhibitionrates of the aqueous extract, 30% ethanol extract, 50% ethanol ex-tract, 70% ethanol extract, 100% ethanol extract were 35.90%,79.03%, 87.72%, 84.97%, 10.33% at the seventh day, respectively.The differences in the antioxidant activities among all the extractswere significant (P < 0.05). 50% ethanol extract and 70% ethanol ex-tract could extend the induction period of linoleic acid autoxida-tion significantly. In the process of inhibiting linoleic acidperoxidation, the antioxidant components in the extract playedthe combined effect through the various antioxidant mechanisms,such as scavenging free radicals, binding metal catalysts, decom-posing peroxides, breaking reaction chains (Adedapo et al., 2008).

0.00

0.05

0.10

0.15

0.20

0.20 0.04 0.60 0.80 1.00 1.20 1.40Concentration (mg/ml)

Abs

orba

nce

at 7

00nm Aqueous extract

30% Ethanol extract

50% Ethanol extract

70% Ethanol extract

100%Ethanol extract

Fig. 3. Reducing power of DWGEs.

So, the antioxidant performance of the extract in the linoleic acidsystem could overall reflect the antioxidant activity of all compo-nents in the extract.

3.7. Relationship between total phenolic content and antioxidantactivity

In the previous reports on plant extracts (Chew, Goh, & Lim,2009; Liu, Lin, Wang, Chen, & Yang, 2009), it was found that phen-olics were the main antioxidant components and its total contentwas directly proportional to the antioxidant activity. But in this pa-per, the relationship between the total phenolic content and theantioxidant activities of the extracts were complex. Several reasonscould be provided for this observation: (1) the different extractionsolvent resulted in the differences of the extracts in their composi-tions, and consequently their antioxidant activities (Pinelo,Manzocco, Nunez, & Nicoli, 2004); (2) the antioxidant assays usedwere based on different mechanisms and conditions, so that theymay present differing results, each only partially reflecting theantioxidant activity (Arabshahi-Delouee & Urooj, 2007; Delgado-Andrade et al., 2005; Hseu et al., 2008); (3) the FCR method to mea-sure the total phenolic content of extracts could be disturbed byother components (Perez-Jimenez & Saura-Calixto, 2005; Prior,Wu, & Schaich, 2005). It was also thought that all other solublecompounds present in the extracts, including proteins, peptides,polysaccharides, and pigments, could be responsible for the antiox-idant activity partly (Prior et al., 2005).

4. Conclusions

In this study, all the DWGEs exhibited antioxidant activities inthe DPPH radical scavenging assay, the ABTS radical scavenging as-say, the reducing power assay, and inhibition of linoleic acid perox-idation. The yields of the aqueous and aqueous ethanol extractswere acceptable, but the antioxidant activity of aqueous extractwas low. Among the various extracts, all aqueous ethanol extractsand the ethanol extract showed high antioxidant activity, while theethanol extract was low in yield. Considering both the yield andthe antioxidant activity, the 50% ethanol extract and the 70% etha-nol extract were optimal. The total phenolic contents of the ex-tracts were inconsistent with the antioxidant activities of theextracts, pending further analysis of their specific composition toexplain the underlying mechanism. It is indicated by the resultsof this work that wheat germ, on the condition that proper solventand extraction processes are established, could serve as a source ofnatural antioxidants or nutraceuticals.

Acknowledgements

This work was supported by the Fundamental Research Fundsfor the Central Universities (JUSRP10922) and the AgriculturalKey Technology R & D Program of Jiangsu (BE 2009361-1).

1126 K.-X. Zhu et al. / Food Chemistry 126 (2011) 1122–1126

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