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Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase
Process Biochemistry 41 (2006) 1296–1302 www.elsevier.com/locate/procbio
Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase Kexue Zhu a,b, Huiming Zhou a,b,*, Haifeng Qian a a
School of Food Science and Technology, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036 Jiangsu Province, PR China b Key Laboratory of Food Science and Safety, Ministry of Education, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036 Jiangsu Province, PR China Received 9 August 2005; received in revised form 24 October 2005; accepted 23 December 2005
Abstract Wheat germ protein hydrolysates (WGPH) were obtained by enzymatic hydrolysis of defatted wheat germ protein isolates using Alcalase 2.4L FG. The degree of hydrolysis (DH) of WGPH was determined to be about 25% using pH-stat method. The molecular mass distribution of WGPH was lower than 1500 Da. The antioxidant and free radical-scavenging activities of WGPH were investigated by employing several in vitro assay systems, including the linoleic acid emulsion model system, 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radical-scavenging, reducing power, and ferrous ion-chelating activity. The antioxidant activity of WGPH was close to that of a-tocopherol in a linoleic acid emulsion system. WGPH showed scavenging activity against free radicals such as DPPH, superoxide, and hydroxyl radicals. The radical-scavenging effect was in a dose-dependent manner, and the EC50 values for DPPH, superoxide, and hydroxyl radicals were found to be 1.30, 0.40 and 0.12 mg/mL, respectively. Moreover, WGPH also exhibited notable reducing power and strong chelating effect on Fe2+. The data obtained by the in vitro systems obviously established the antioxidant potency of WGPH. # 2006 Elsevier Ltd. All rights reserved. Keywords: Wheat germ protein hydrolysates; Antioxidant activity; Free radical-scavenging activity; DPPH; Alcalase
1. Introduction Raw wheat germ, containing as much as 10% oil, is mainly used in food, medical and cosmetic industries as a source of oil [1]. The main by-product of the oil extraction process is a defatted wheat germ meal, which has relatively high protein content (30%) [2], making the defatted wheat germ meal as one of the most attractive and promising sources of vegetable proteins. Annually, there is a rich production of wheat germ in the world. However, the precious wheat germ source has a poor utility for human applications, while the majority is used for animal feeding purposes. Thus, it is for this reason that efforts are being made to devise efficient methods for the recovery of proteins from defatted wheat germ meal and to prepare acceptable products for human consumption. One of the possible ways to utilize wheat germ proteins is to produce
* Corresponding author. Tel.: +86 510 5913539; fax: +86 510 5913539. E-mail addresses: [email protected] (K. Zhu), [email protected] (H. Zhou). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.12.029
protein isolates by alkaline extraction and subsequent isoelectric precipitation—a procedure similar to soy protein isolates extraction [2]. However, wheat germ protein isolates have lower functional properties compared to soy and casein protein products [3]. Enzymatic hydrolysis is widely applied to improve and upgrade the functional and nutritional properties of food proteins. Claver and Zhou [4] reported that the recovery yield and functional properties of defatted wheat germ protein can be improved by various food grade proteases treatment, such as Alcalase, Flavourzyme, Papain, Neutrase, and Protamex. Their results also showed that Alcalase 2.4L was the most effective of the five proteases used. Pepsin and trypsin were reported to be able to improve the emulsifying activity and emulsifying stability of wheat germ protein [5]. In addition, various physiological activities have been detected in the hydrolysates derived from the proteolytic hydrolysis of many food proteins, such as antimicrobial, immunomodulatory, antihypertensive, antioxidant, opioide and mineral binding [6]. Several angiotensin I-converting enzyme inhibitory peptides from wheat germ hydrolyzate were reported by Matsui et al. [7]. In recent years, there is a growing interest to identify antioxidative properties in many natural sources
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including some dietary protein compounds due to the potential health hazards of some synthetic antioxidants. Up to now, numerous peptides derived from hydrolyzed food proteins have been shown to have noteworthy antioxidative activities against the peroxidation of lipids or fatty acids [8]. However, little information about the antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates was available until now. The present study reports on the antioxidant and free radicalscavenging activities of wheat germ protein hydrolysates (WGPH) prepared with Alcalase 2.4L FG. Different measurements, including the ability to inhibit the autoxidation of linoleic acid, the scavenging effect on free radical, reducing power, and ferrous ion-chelating activity are used to evaluate the antioxidant activities. 2. Materials and methods 2.1. Materials and chemicals Raw wheat germ (RWG) was donated by Huaian Xinfeng Flour Mill (Jiangsu, China). Linoleic acid, a,a-diphenyl-b-picrylhydrazyl (DPPH), 2deoxy-D-ribose, nitroblue tetrazolium salt, xanthine, xanthine oxidase (from buttermilk, 0.049 U/mL), a-tocopherol, ferrozine, butylated hydroxytoluene (BHT), and ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, USA). Alcalase 2.4L FG was acquired from Novo Co. (Novo Nordisk, Bagsvaerd, Denmark). All other chemicals used in the experiments were of analytical grade.
2.2. Defatted wheat germ flours (DWGF) preparation Raw wheat germ was selected and cleaned to remove contaminants. The enzymes of wheat germ were inactivated by heating for 20 min at 105 8C and it was then defatted with n-hexane for 8 h and air-dried at room temperature. The defatted wheat germ meal was milled using a laboratory scale hammer mill. The resulting flour (DWGF) was sieved through a 60-mesh screen and was kept in sealed glass jars at 4 8C until used.
2.3. Protein isolates preparation Defatted wheat germ protein isolates were prepared according to the process described by Hettiarachchy et al. [3] with minor modifications. DWGF was dispersed in 1.0 mol/L NaCl solution (1:8, w/v) and stirred for 30 min at ambient temperature, and then its pH was adjusted to 9.5 by using 1 mol/L NaOH. After stirring for 30 min, the suspension was centrifuged at 8000 rpm for 20 min at 4 8C. The supernatant was adjusted to pH 4.0 with 1.0 mol/L HCl to precipitate the proteins, and centrifuged again at 8000 rpm for 20 min at 4 8C. The precipitates were washed several times with distilled water (pH 4.0), dispersed in a small amount of distilled water, and adjusted to pH 7.0 by using 0.1 mol/L NaOH. The dispersed product was freeze-dried.
2.4. Preparation of WGPH The 10% (w/v) protein isolates solution was prepared and hydrolyzed with Alcalase for 6 h. The hydrolysis was carried out using the following hydrolysis parameters: enzyme–substrate ratio (E/S) = 0.4 AU/g of protein; temperature (T) = 50 8C; pH 8.0. The hydrolysis was conducted in a 200 mL reaction vessel, equipped with a stirrer, thermometer, and pH electrode. Hydrolysis was stopped by heat treatment at 90 8C for 10 min. Hydrolysates were clarified by centrifuging at 3000 rpm for 20 min to remove insoluble substrate fragments and residual enzyme. The hydrolysates were then frozen, lyophilized and stored at 20 8C before further analysis.
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2.5. Degree of hydrolysis The degree of hydrolysis, defined as the percentage of number of peptide bonds cleaved divided by the total number of peptide bonds in a protein, was calculated from the consumption of base (NaOH) by the pH-stat method of Adler-Nissen [9]. The percent DH was calculated by the following equation: DHð%Þ ¼
h BNb 100 ¼ 100 htot aMp htot
where B and Nb represent the amount of NaOH consumed during the proteolysis of the substrate and its normality, respectively, a the average degree of dissociation of the a-NH2 groups in the protein substrate (0.874 for 50 8C and pH 8.0), Mp the mass (g) of the protein (N 5.7), and htot is the total number of peptide bonds available for proteolytic hydrolysis (7.8 m equiv./g).
2.6. Molecular weight distribution profile Molecular weight distributions of WGPH were determined by gel permeation chromatography (GPC) using a high-performance liquid chromatography (HPLC) system (Waters 600, USA). A TSK gel2000 SWXL column (7.8 i.d. 300 mm, Tosoh, Tokyo, Japan) was equilibrated with 45% acetonitrile (v/v) in the presence of 0.1% trifluoroacetic acid. The hydrolysates (100 mg/ 50 mL) were applied to the column and eluted at a flow rate of 0.5 mL/min and monitored at 220 nm at room temperature. A molecular weight calibration curve was prepared from the average retention time of the following standards: cytochrome C (12,500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide GGYR (451 Da), and tripeptide GGG (189 Da) (Sigma Co., St. Louis, MO, USA).
2.7. Inhibition of linoleic acid autoxidation The antioxidative activity of WGPH with different periods of incubation was measured in a linoleic acid model system according to the methods of Osawa and Namiki [10] with some modifications. Each sample (10 mg) was dissolved in 10 mL 50 mM phosphate buffer (pH 7.0), and added to a solution of 0.15 mL linoleic acid and 10 mL 99.5% ethanol. Then, the total volume was adjusted to 25 mL with distilled water. The mixture was incubated in a conical flask with a screw cap at 40 1 8C in a dark room, and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The ferric thiocyanate value was measured according to the method of Mitsuda et al. [11]. The reaction solution (100 mL) incubated in the linoleic acid model system described herein was mixed with 4.7 mL 75% ethanol, 0.1 mL 30% ammonium thiocyanate, and 0.1 mL 0.02 M ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following colour development with FeCl2 and thiocyanate at different intervals during the incubation period at 40 1 8C.
2.8. Amino acid analysis For the determination of the amino acids, samples of WGPH and protein isolates (150 mg) were subjected to acid hydrolysis with 5 mL of 6 mol/L HCl under nitrogen atmosphere for 24 h at 110 8C. Each hydrolyzate was washed into a 50 mL volumetric flask and made up to the mark with distilled water. The amino acids were subjected to RP-HPLC analysis (Agilent1100, USA) after precolumn derivatization with o-phthaldialdehyde (OPA) [12] or with 9-fluorenylmethyl chloroformate (FMOC) [13]. Methionine and cysteine were determined separately as their oxidation products according to the performic acid procedure of Moore [14] prior to hydrolysis in 6N HCl. Amino acid composition was reported as g amino acid per 100 g protein.
2.9. Scavenging effect on DPPH free radical The scavenging effect of WGPH on a,a-diphenyl-b-picrylhydrazyl (DPPH) free radical was measured according to the method of Shimada et al. [15] with some modifications. A volume of 2 mL of each sample was added to 2 mL of 0.1 mM DPPH in 95% ethanol. The mixture was shaken and left for 60 min at
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room temperature, and the absorbance of the resulting solution was measured at 517 nm. Triplicate tests were conducted for each sample. A lower absorbance represented a higher DPPH scavenging activity. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
2 mM FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the mixture was shaken vigorously and left standing at room temperature for 10 min. The absorbance of the solution was then measured spectrophotometrically at 562 nm. EDTA was used as a positive control. All tests and analyses were carried out in triplicate. The percentage of inhibition of ferrozine–Fe2+ complex formation was given in the formula: ferrous ion chelating activity ¼ ½ðA0 A1 Þ=A0 100 where A0 was the absorbance of the control and A1 was the absorbance in the presence of samples.
2.10. Determination of superoxide radical-scavenging activity The scavenging activity of WGPH was determined using the nitro-blue tetrazolium (NBT) reduction method. In this method, O2 generated in vitro by the xanthine oxidase reduced the yellow dye (NBT2+) to produce the blue formazan, which was measured spectrophotometrically at 560 nm [16]. The capacity of the hydrolysates to scavenge the superoxide radicals was assayed as follows: the reaction mixture contained 0.5 mL of 0.8 mM xanthine in 0.1 mM phosphate buffer (pH 8.0), 0.48 mM NBT in 0.1 mM phosphate buffer (pH 8.0) and 0.1 mL of the sample solution. After heating at 37 8C for 10 min, the reaction was initiated by adding 1.0 mL of XOD (0.049 U/mL) and carried out at 37 8C for 20 min, the reaction was stopped by adding 2.0 mL of 69 mM SDS. The absorbance of the reaction mixture was measured at 560 nm. Triplicate tests were conducted for each sample. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
2.11. Hydroxyl radical-scavenging activity The effect of hydroxyl radicals was assayed using the 2-deoxy-D-ribose oxidation method of Halliwell et al. [17]. The following reagents were added into a reaction tube in the following order: 0.4 mL of KH2PO4–KOH buffer (pH 7.5), 0.1 mL sample solution of various concentrations, and 0.1 mL of 1 mM EDTA, 10 mM H2O2, 60 mM 2-deoxy-D-ribose, 2 mM ascorbic acid, and 1 mM FeCl3. Solutions of FeCl3, H2O2, and ascorbic acid were made just before use. The reaction solution was incubated at 37 8C for 1 h. Then 1 mL of 20% (v/v) trichloroacetic acid was added to stop the reaction. The colour was developed by adding 1 mL of 1% TBA (w/v) into reaction tubes, which were placed into a temperature-controlled water bath at 100 8C for 15 min. The tubes were cooled in ice, and then the absorbance was measured at 532 nm. All values were determined in three replicates. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
3. Results and discussion 3.1. Enzymatic hydrolysis of wheat germ protein isolates Alcalase is an alkaline protease that has been used not only for production of protein hydrolysates with better functional and nutritional characteristics than the original proteins, but also for the generation of bioactive peptides. Claver and Zhou [4] reported that Alcalase can solubilize 85% wheat germ proteins with the desirable functional properties. Wheat germ proteins treated by Alcalase 2.4L were the most favorable in producing potent ACE inhibitors [7]. The hydrolysis of defatted wheat germ protein isolates with Alcalase was monitored for 6 h by pH-stat method. Fig. 1 shows a typical hydrolysis curve obtained under experimental conditions, resulting in an increase in DH as a function of reaction time. The curve showed a high rate of hydrolysis for the first 1 h, and DH reached about 25% after 6 h of hydrolysis. These results agree with the earlier report of Claver and Zhou [4]. The molecular weight distribution of hydrolysates is presented in Fig. 2. The hydrolysates treated by Alcalase for 3, 6 and 12 h showed similar molecular weight distribution (data not shown), which indicated that prolonging the reaction time did not produce any significant improvement in the DH after 3 h. The molecular mass of the main peaks of the hydrolysates was lower than 1500 Da (range from 1050 to 180 Da). In addition, the antioxidant activities of various hydrolysates prepared at different hydrolytic periods were investigated and the antioxidant activities of the 6 h hydrolysates were found to be high (data not shown). Therefore, the 6 h hydrolysate was selected to determine antioxidant and free radical-scavenging activities of the peptide mixtures.
2.12. Reducing power The reducing power of WGPH was measured according to the method of Oyaizu [18]. Sample (10, 20, 30 and 40 mg) was added to 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 8C for 20 min. Then 2.5 mL of 10% TCA was added to the reaction mixture. A volume of 2.5 mL from each incubated mixture was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride in a test tube. After a 10 min reaction time, the absorbance of the resulting solution was measured at 700 nm. Triplicate tests were conducted for each sample. Increased absorbance of the reaction mixture indicated increased reducing power.
2.13. Determination of ferrous ion-chelating activity Ferrous ion-chelating activity was determined according to the method of Dinis et al. [19]. The hydrolysate samples (3 mL) were added to a solution of
Fig. 1. Hydrolysis curve of defatted wheat germ protein isolates treated with Alcalase 2.4L FG. Reaction conditions: pH, 8.0; temperature, 50 8C; enzyme– substrate ratio, 0.4 AU/g of protein; substrate concentration, 10% (w/v).
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Table 1 Comparative amino acid profile of WGPH and wheat germ protein isolates (g/ 100 g protein) Amino acid
Protein isolates
WGPH
Asp Glub Ser His Gly Thr Arg Ala Tyr Cys Val Met Phe Ile Leu Lys Pro Trp
8.88 15.32 4.86 3.13 6.18 4.12 9.47 5.80 3.24 0.56 6.65 2.11 5.08 4.52 7.81 7.07 4.63 n.dc
8.27 14.71 4.45 2.48 8.67 4.17 8.71 6.98 3.08 0.53 6.09 2.16 4.53 4.22 7.05 7.61 5.96 n.d c
Total hydrophobic amino acidsd
42.78
45.67
a
Fig. 2. The molecular weight distribution profiles of WGPH. GPC-HPLC conditions: column, TSK G2000 SWXL column (7.8 i.d. 300 mm, Tosoh); sample, 100 mg/50 mL; mobile phase, 45% acetonitrile/water/trifluoroacetic acid (45/55/0.1, v/v/v); flow rate, 0.5 mL/min; detection, absorbance at 220 nm. The arrows indicate the elution times of molecular mass markers.
a
3.2. Antioxidant activities of WGPH
b c d
The antioxidative activities of WGPH were measured in linoleic acid emulsion system and compared with those of atocopherol and BHT. As shown in Fig. 3, the autoxidation of linoleic acid was effectively inhibited by the addition of WGPH. The antioxidative activity of WGPH was close to that of a-tocopherol, while it was much lower than that of BHT. Therefore, the result indicated that WGPH seemed to contain some antioxidative peptides and amino acids. The amino acid compositions of wheat germ protein isolates and WGPH were analyzed and shown in Table 1. Although Glu, Arg and Asp were the major constituent amino acids of both protein isolates and WGPH, the amino acid composition of WGPH was different from that of protein isolates. The contents of Gly, Lys, Ala and Pro in WGPH were much higher than those of protein isolates. In addition, the total content of hydrophobic amino acids of WGPH was
Aspartic acid + asparagine. Glutamic acid + glutamine. Not determined. Csontaining Gly, Ala, Val, Leu, Pro, Met, Phe, Trp and Ile.
higher than that of protein isolates. For protein hydrolysates and peptides, an increase in hydrophobicity will increase their solubility in lipid and therefore enhances their antioxidative activity [20,21]. Six peptides from digests of a soybean protein were reported to exhibit high antioxidative activities against lipid peroxidation [22]. These peptides were composed of 5– 16 amino acid residues, including hydrophobic amino acids, Val or Leu, at the N-terminal positions, and Pro, His, or Tyr in the sequences. Chen et al. [23] reported that the N-terminal histidine could contribute higher antioxidative activity to the peptides. In addition, several amino acids, such as Tyr, Met, His, Lys, and Trp, are generally accepted as antioxidants in spite of their pro-oxidative effects in some cases [24]. These information seem to be important in explaining how the hydrolysates possess their antioxidative activity. 3.3. Scavenging effect on DPPH free radical
Fig. 3. The antioxidant activity of WGPH. WGPH was incubated in a linoleic acid emulsion system for 6 days as described in Section 2. The degree of linoleic acid oxidation was measured by the ferric thiocyanate method at every 24 h interval. Butylated hydroxytoluene and a-tocopherol were used as positive controls.
The DPPH free radicals, which are stable in ethanol, show maximum absorbance at 517 nm. When DPPH radicals encounter a proton-donating substance such as an antioxidant, the radicals would be scavenged and the absorbance is reduced. Thus, the DPPH radicals were widely used to investigate the scavenging activity of some natural compounds. Fig. 4 shows the results of scavenging DPPH radical ability of WGPH at various concentrations. WGPH showed dose-dependent DPPH radicals scavenging activity. The EC50 (meaning the concentration that causes a decrease in the initial DPPH concentration by 50%) is a parameter widely used to measure the antiradical efficiency. The lower the EC50, the higher the free radicalscavenging ability. The EC50 value of WGPH for scavenging
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Fig. 4. Scavenging effect on DPPH free radical of different concentrations of WGPH. Butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls to compare the scavenging effect of WGPH. Regression equation was obtained from linear regression of the concentrations of WGPH and DPPH radical-scavenging effects. Each value is expressed as mean S.D. (n = 3).
activity against DPPH was calculated from the regression equation to be 1.30 mg/mL. The result showed that WGPH is a free radical inhibitor, as well as a primary antioxidant that reacts with free radicals, which may limit the occurrence of free radical damage in human body. 3.4. Superoxide radical-scavenging activity Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxidative species, such as single oxygen and hydroxyl radicals [25]. Furthermore, superoxide radical is considered to play an important role in the peroxidation of lipids [26]. Therefore, studying the scavenging effects of WGPH on superoxide radicals is one of the most important ways of clarifying the mechanism of antioxidant activity. The superoxide radical-scavenging activity of WGPH was determined using the cellular xanthine/xanthine oxidase system as a superoxide source. The result is presented in Fig. 5. The scavenging activity of WGPH on superoxide radicals increased with increasing concentrations. The EC50 value of WGPH for superoxide radicals was 0.40 mg/mL. These results indicated that WGPH had a notable effect on scavenging of superoxide radicals. 3.5. Hydroxyl radical-scavenging activity
Fig. 6. Hydroxyl radical-scavenging activities of different concentrations of WGPH by the deoxyribose method. Butylated hydroxytoluene (BHT) was used as positive control. Regression equation was obtained from linear regression of the concentrations of WGPH and hydroxyl radical-scavenging effects. Each value is expressed as mean S.D. (n = 3).
damage and hence human diseases [27]. Among the oxygen radicals specifically, the hydroxyl radical is the most reactive and severely damages adjacent biomolecules such as all proteins, DNA, PUFA, nucleic acid, and almost any biological molecule it touches. This damage causes aging, cancer and several diseases [28]. Therefore, the removal of hydroxyl radical is probably one of the most effective defenses of a living body against various diseases. The scavenging effect against hydroxyl radicals was investigated by using the 2-deoxyribose oxidation method. Fig. 6 shows the hydroxyl radical-scavenging effects of WGPH. The scavenging effect of WGPH on hydroxyl radicals was concentration-dependent. WGPH at the final concentration of 0.16 mg/mL exhibited 67.8% scavenging effect on hydroxyl radical, and the EC50 value was 0.12 mg/mL. According to the present findings, WGPH might be used to provide a good hydroxyl radical scavenger for humans and foods. 3.6. Reducing power For the measurements of the reductive ability, we investigated the Fe3+–Fe2+ transformation in the presence of WGPH using the method of Oyaizu [18]. The reducing power of WGPH was excellent and increased steadily with increasing amount of sample (Fig. 7). At 40 mg, the reducing power was higher than 1.45. The reducing power of bioactive compounds
The reactive oxygen radicals are unstable, and react readily with other groups or substances in the body, resulting in cell
Fig. 5. Superoxide radical-scavenging activities of different concentrations of WGPH in the cellular xanthine/xanthine oxidase system by the NBT method. Each value is expressed as mean S.D. (n = 3).
Fig. 7. Reducing power of different amounts of WGPH using spectrophotometric detection of the Fe3+–Fe2+ transformations. Regression equation was obtained from linear regression of the amounts of WGPH and the absorbances at 700 nm. Each value is expressed as mean S.D. (n = 3).
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References
Fig. 8. Ferrous ion-chelating activities of different concentrations of WGPH. EDTA was used as positive control. Each value is expressed as mean S.D. (n = 3).
had been reported to be associated with their antioxidant activity [29,30]. Thus, a relation should be located between reducing power and the antioxidant effect. Our data on the reducing power of WGPH suggested that it was likely to contribute significantly towards the observed antioxidant effect. 3.7. Ferrous ion-chelating activity The chelating of ferrous ions by WGPH was estimated by the method of Dinis et al. [19]. Ferrozine can quantitatively form complexes with Fe2+ ion. In the presence of chelating agents, the complex formation is disrupted resulting to a decrease in the red colour of the complex. Measurement of colour reduction therefore makes possible the estimation of the metal chelating activity of the coexisting chelator [31]. In this assay, WGPH as well as EDTA interfered with the formation of ferrous and ferrozine complex, thus suggesting that it has a chelating activity and captures ferrous ion before ferrozine. EDTA is a known metal ion chelator; and therefore the chelating effect of WGPH was compared with it. The ferrous ion-chelating effects of WGPH and EDTA are shown in Fig. 8. WGPH showed a strong chelating activity on Fe2+ ion. It displayed 89.0% chelating effect on Fe2+ ion at a concentration of 1000 mg/mL. However, the chelating ability was slightly lower than that of EDTA. This indicated that WGPH had an effective capacity for iron binding, suggesting that its action as an antioxidant may be related to its iron binding capacity. 4. Conclusion The enzymatic hydrolysis of wheat germ protein through the action of Alcalase 2.4L FG provided a high proportion of peptides from 1500 Da to free amino acids. Freeze-dried WGPH showed a relatively higher antioxidant activity and free radical-scavenging activities. At present, consumer demand for natural functional foods, has been increasing, and therefore WGPH will be used as a functional food ingredient in pharmaceutical and food industries in the future. However, further detailed studies on WGPH and its peptide fractions with regard to antioxidant activity in vivo and the different antioxidant mechanisms are needed.
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[24] Chen HM, Muramoto K, Yamauchi F, Nokihara K. Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J Agric Food Chem 1996;44:2619–23. [25] Aurand LW, Boonme NH, Gidding GG. Superoxide and singlet oxygen in milk lipid peroxidation. J Dairy Sci 1977;60:363–9. [26] Dahl MK, Richardson T. Photogeneration of superoxide anion in serum of bovine milk and in model systems containing riboflavin and amino acids. J Dairy Sci 1978;61:400–7. [27] Halliwell B, Gutteridge JMC. Free radicals in biology and medicine, 2nd ed., Oxford: Clarendon Press; 1989. p. 1–20.
[28] Aruoma OI. Free radicals oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc 1998;75:199–211. [29] Meir S, Kanner J, Akiri B, Hadas SP. Determination and involvement of aqueous reducing compounds in oxidative defense systems of various senescing leaves. J Agric Food Chem 1995;43:1813–9. [30] Juntachote T, Berghofer E. Antioxidative properties and stability of ethanolic extracts of holy basil and Galangal. Food Chem 2005;92:193–202. [31] Yamaguchi F, Ariga T, Yoshimira Y, Nakazawa H. Antioxidant and antiglycation of carcinol from Garcinia indica fruit rind. J Agric Food Chem 2000;48:180–5.
Antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates (WGPH) prepared with alcalase Kexue Zhu a,b, Huiming Zhou a,b,*, Haifeng Qian a a
School of Food Science and Technology, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036 Jiangsu Province, PR China b Key Laboratory of Food Science and Safety, Ministry of Education, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036 Jiangsu Province, PR China Received 9 August 2005; received in revised form 24 October 2005; accepted 23 December 2005
Abstract Wheat germ protein hydrolysates (WGPH) were obtained by enzymatic hydrolysis of defatted wheat germ protein isolates using Alcalase 2.4L FG. The degree of hydrolysis (DH) of WGPH was determined to be about 25% using pH-stat method. The molecular mass distribution of WGPH was lower than 1500 Da. The antioxidant and free radical-scavenging activities of WGPH were investigated by employing several in vitro assay systems, including the linoleic acid emulsion model system, 1,1-diphenyl-2-picrylhydrazyl (DPPH)/superoxide/hydroxyl radical-scavenging, reducing power, and ferrous ion-chelating activity. The antioxidant activity of WGPH was close to that of a-tocopherol in a linoleic acid emulsion system. WGPH showed scavenging activity against free radicals such as DPPH, superoxide, and hydroxyl radicals. The radical-scavenging effect was in a dose-dependent manner, and the EC50 values for DPPH, superoxide, and hydroxyl radicals were found to be 1.30, 0.40 and 0.12 mg/mL, respectively. Moreover, WGPH also exhibited notable reducing power and strong chelating effect on Fe2+. The data obtained by the in vitro systems obviously established the antioxidant potency of WGPH. # 2006 Elsevier Ltd. All rights reserved. Keywords: Wheat germ protein hydrolysates; Antioxidant activity; Free radical-scavenging activity; DPPH; Alcalase
1. Introduction Raw wheat germ, containing as much as 10% oil, is mainly used in food, medical and cosmetic industries as a source of oil [1]. The main by-product of the oil extraction process is a defatted wheat germ meal, which has relatively high protein content (30%) [2], making the defatted wheat germ meal as one of the most attractive and promising sources of vegetable proteins. Annually, there is a rich production of wheat germ in the world. However, the precious wheat germ source has a poor utility for human applications, while the majority is used for animal feeding purposes. Thus, it is for this reason that efforts are being made to devise efficient methods for the recovery of proteins from defatted wheat germ meal and to prepare acceptable products for human consumption. One of the possible ways to utilize wheat germ proteins is to produce
* Corresponding author. Tel.: +86 510 5913539; fax: +86 510 5913539. E-mail addresses: [email protected] (K. Zhu), [email protected] (H. Zhou). 1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.12.029
protein isolates by alkaline extraction and subsequent isoelectric precipitation—a procedure similar to soy protein isolates extraction [2]. However, wheat germ protein isolates have lower functional properties compared to soy and casein protein products [3]. Enzymatic hydrolysis is widely applied to improve and upgrade the functional and nutritional properties of food proteins. Claver and Zhou [4] reported that the recovery yield and functional properties of defatted wheat germ protein can be improved by various food grade proteases treatment, such as Alcalase, Flavourzyme, Papain, Neutrase, and Protamex. Their results also showed that Alcalase 2.4L was the most effective of the five proteases used. Pepsin and trypsin were reported to be able to improve the emulsifying activity and emulsifying stability of wheat germ protein [5]. In addition, various physiological activities have been detected in the hydrolysates derived from the proteolytic hydrolysis of many food proteins, such as antimicrobial, immunomodulatory, antihypertensive, antioxidant, opioide and mineral binding [6]. Several angiotensin I-converting enzyme inhibitory peptides from wheat germ hydrolyzate were reported by Matsui et al. [7]. In recent years, there is a growing interest to identify antioxidative properties in many natural sources
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including some dietary protein compounds due to the potential health hazards of some synthetic antioxidants. Up to now, numerous peptides derived from hydrolyzed food proteins have been shown to have noteworthy antioxidative activities against the peroxidation of lipids or fatty acids [8]. However, little information about the antioxidant and free radical-scavenging activities of wheat germ protein hydrolysates was available until now. The present study reports on the antioxidant and free radicalscavenging activities of wheat germ protein hydrolysates (WGPH) prepared with Alcalase 2.4L FG. Different measurements, including the ability to inhibit the autoxidation of linoleic acid, the scavenging effect on free radical, reducing power, and ferrous ion-chelating activity are used to evaluate the antioxidant activities. 2. Materials and methods 2.1. Materials and chemicals Raw wheat germ (RWG) was donated by Huaian Xinfeng Flour Mill (Jiangsu, China). Linoleic acid, a,a-diphenyl-b-picrylhydrazyl (DPPH), 2deoxy-D-ribose, nitroblue tetrazolium salt, xanthine, xanthine oxidase (from buttermilk, 0.049 U/mL), a-tocopherol, ferrozine, butylated hydroxytoluene (BHT), and ascorbic acid were purchased from Sigma Chemical Co. (St. Louis, USA). Alcalase 2.4L FG was acquired from Novo Co. (Novo Nordisk, Bagsvaerd, Denmark). All other chemicals used in the experiments were of analytical grade.
2.2. Defatted wheat germ flours (DWGF) preparation Raw wheat germ was selected and cleaned to remove contaminants. The enzymes of wheat germ were inactivated by heating for 20 min at 105 8C and it was then defatted with n-hexane for 8 h and air-dried at room temperature. The defatted wheat germ meal was milled using a laboratory scale hammer mill. The resulting flour (DWGF) was sieved through a 60-mesh screen and was kept in sealed glass jars at 4 8C until used.
2.3. Protein isolates preparation Defatted wheat germ protein isolates were prepared according to the process described by Hettiarachchy et al. [3] with minor modifications. DWGF was dispersed in 1.0 mol/L NaCl solution (1:8, w/v) and stirred for 30 min at ambient temperature, and then its pH was adjusted to 9.5 by using 1 mol/L NaOH. After stirring for 30 min, the suspension was centrifuged at 8000 rpm for 20 min at 4 8C. The supernatant was adjusted to pH 4.0 with 1.0 mol/L HCl to precipitate the proteins, and centrifuged again at 8000 rpm for 20 min at 4 8C. The precipitates were washed several times with distilled water (pH 4.0), dispersed in a small amount of distilled water, and adjusted to pH 7.0 by using 0.1 mol/L NaOH. The dispersed product was freeze-dried.
2.4. Preparation of WGPH The 10% (w/v) protein isolates solution was prepared and hydrolyzed with Alcalase for 6 h. The hydrolysis was carried out using the following hydrolysis parameters: enzyme–substrate ratio (E/S) = 0.4 AU/g of protein; temperature (T) = 50 8C; pH 8.0. The hydrolysis was conducted in a 200 mL reaction vessel, equipped with a stirrer, thermometer, and pH electrode. Hydrolysis was stopped by heat treatment at 90 8C for 10 min. Hydrolysates were clarified by centrifuging at 3000 rpm for 20 min to remove insoluble substrate fragments and residual enzyme. The hydrolysates were then frozen, lyophilized and stored at 20 8C before further analysis.
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2.5. Degree of hydrolysis The degree of hydrolysis, defined as the percentage of number of peptide bonds cleaved divided by the total number of peptide bonds in a protein, was calculated from the consumption of base (NaOH) by the pH-stat method of Adler-Nissen [9]. The percent DH was calculated by the following equation: DHð%Þ ¼
h BNb 100 ¼ 100 htot aMp htot
where B and Nb represent the amount of NaOH consumed during the proteolysis of the substrate and its normality, respectively, a the average degree of dissociation of the a-NH2 groups in the protein substrate (0.874 for 50 8C and pH 8.0), Mp the mass (g) of the protein (N 5.7), and htot is the total number of peptide bonds available for proteolytic hydrolysis (7.8 m equiv./g).
2.6. Molecular weight distribution profile Molecular weight distributions of WGPH were determined by gel permeation chromatography (GPC) using a high-performance liquid chromatography (HPLC) system (Waters 600, USA). A TSK gel2000 SWXL column (7.8 i.d. 300 mm, Tosoh, Tokyo, Japan) was equilibrated with 45% acetonitrile (v/v) in the presence of 0.1% trifluoroacetic acid. The hydrolysates (100 mg/ 50 mL) were applied to the column and eluted at a flow rate of 0.5 mL/min and monitored at 220 nm at room temperature. A molecular weight calibration curve was prepared from the average retention time of the following standards: cytochrome C (12,500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide GGYR (451 Da), and tripeptide GGG (189 Da) (Sigma Co., St. Louis, MO, USA).
2.7. Inhibition of linoleic acid autoxidation The antioxidative activity of WGPH with different periods of incubation was measured in a linoleic acid model system according to the methods of Osawa and Namiki [10] with some modifications. Each sample (10 mg) was dissolved in 10 mL 50 mM phosphate buffer (pH 7.0), and added to a solution of 0.15 mL linoleic acid and 10 mL 99.5% ethanol. Then, the total volume was adjusted to 25 mL with distilled water. The mixture was incubated in a conical flask with a screw cap at 40 1 8C in a dark room, and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The ferric thiocyanate value was measured according to the method of Mitsuda et al. [11]. The reaction solution (100 mL) incubated in the linoleic acid model system described herein was mixed with 4.7 mL 75% ethanol, 0.1 mL 30% ammonium thiocyanate, and 0.1 mL 0.02 M ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following colour development with FeCl2 and thiocyanate at different intervals during the incubation period at 40 1 8C.
2.8. Amino acid analysis For the determination of the amino acids, samples of WGPH and protein isolates (150 mg) were subjected to acid hydrolysis with 5 mL of 6 mol/L HCl under nitrogen atmosphere for 24 h at 110 8C. Each hydrolyzate was washed into a 50 mL volumetric flask and made up to the mark with distilled water. The amino acids were subjected to RP-HPLC analysis (Agilent1100, USA) after precolumn derivatization with o-phthaldialdehyde (OPA) [12] or with 9-fluorenylmethyl chloroformate (FMOC) [13]. Methionine and cysteine were determined separately as their oxidation products according to the performic acid procedure of Moore [14] prior to hydrolysis in 6N HCl. Amino acid composition was reported as g amino acid per 100 g protein.
2.9. Scavenging effect on DPPH free radical The scavenging effect of WGPH on a,a-diphenyl-b-picrylhydrazyl (DPPH) free radical was measured according to the method of Shimada et al. [15] with some modifications. A volume of 2 mL of each sample was added to 2 mL of 0.1 mM DPPH in 95% ethanol. The mixture was shaken and left for 60 min at
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room temperature, and the absorbance of the resulting solution was measured at 517 nm. Triplicate tests were conducted for each sample. A lower absorbance represented a higher DPPH scavenging activity. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
2 mM FeCl2 (0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL) and the mixture was shaken vigorously and left standing at room temperature for 10 min. The absorbance of the solution was then measured spectrophotometrically at 562 nm. EDTA was used as a positive control. All tests and analyses were carried out in triplicate. The percentage of inhibition of ferrozine–Fe2+ complex formation was given in the formula: ferrous ion chelating activity ¼ ½ðA0 A1 Þ=A0 100 where A0 was the absorbance of the control and A1 was the absorbance in the presence of samples.
2.10. Determination of superoxide radical-scavenging activity The scavenging activity of WGPH was determined using the nitro-blue tetrazolium (NBT) reduction method. In this method, O2 generated in vitro by the xanthine oxidase reduced the yellow dye (NBT2+) to produce the blue formazan, which was measured spectrophotometrically at 560 nm [16]. The capacity of the hydrolysates to scavenge the superoxide radicals was assayed as follows: the reaction mixture contained 0.5 mL of 0.8 mM xanthine in 0.1 mM phosphate buffer (pH 8.0), 0.48 mM NBT in 0.1 mM phosphate buffer (pH 8.0) and 0.1 mL of the sample solution. After heating at 37 8C for 10 min, the reaction was initiated by adding 1.0 mL of XOD (0.049 U/mL) and carried out at 37 8C for 20 min, the reaction was stopped by adding 2.0 mL of 69 mM SDS. The absorbance of the reaction mixture was measured at 560 nm. Triplicate tests were conducted for each sample. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
2.11. Hydroxyl radical-scavenging activity The effect of hydroxyl radicals was assayed using the 2-deoxy-D-ribose oxidation method of Halliwell et al. [17]. The following reagents were added into a reaction tube in the following order: 0.4 mL of KH2PO4–KOH buffer (pH 7.5), 0.1 mL sample solution of various concentrations, and 0.1 mL of 1 mM EDTA, 10 mM H2O2, 60 mM 2-deoxy-D-ribose, 2 mM ascorbic acid, and 1 mM FeCl3. Solutions of FeCl3, H2O2, and ascorbic acid were made just before use. The reaction solution was incubated at 37 8C for 1 h. Then 1 mL of 20% (v/v) trichloroacetic acid was added to stop the reaction. The colour was developed by adding 1 mL of 1% TBA (w/v) into reaction tubes, which were placed into a temperature-controlled water bath at 100 8C for 15 min. The tubes were cooled in ice, and then the absorbance was measured at 532 nm. All values were determined in three replicates. The results were calculated as the percentage of free radical-scavenging effect according to the following formula: scavenging effectð%Þ ¼ ½fðC CBÞ ðS SBÞg=ðC CBÞ 100 where S, SB, C, and CB are the absorbance of the sample, the blank sample, the control, and the blank control, respectively.
3. Results and discussion 3.1. Enzymatic hydrolysis of wheat germ protein isolates Alcalase is an alkaline protease that has been used not only for production of protein hydrolysates with better functional and nutritional characteristics than the original proteins, but also for the generation of bioactive peptides. Claver and Zhou [4] reported that Alcalase can solubilize 85% wheat germ proteins with the desirable functional properties. Wheat germ proteins treated by Alcalase 2.4L were the most favorable in producing potent ACE inhibitors [7]. The hydrolysis of defatted wheat germ protein isolates with Alcalase was monitored for 6 h by pH-stat method. Fig. 1 shows a typical hydrolysis curve obtained under experimental conditions, resulting in an increase in DH as a function of reaction time. The curve showed a high rate of hydrolysis for the first 1 h, and DH reached about 25% after 6 h of hydrolysis. These results agree with the earlier report of Claver and Zhou [4]. The molecular weight distribution of hydrolysates is presented in Fig. 2. The hydrolysates treated by Alcalase for 3, 6 and 12 h showed similar molecular weight distribution (data not shown), which indicated that prolonging the reaction time did not produce any significant improvement in the DH after 3 h. The molecular mass of the main peaks of the hydrolysates was lower than 1500 Da (range from 1050 to 180 Da). In addition, the antioxidant activities of various hydrolysates prepared at different hydrolytic periods were investigated and the antioxidant activities of the 6 h hydrolysates were found to be high (data not shown). Therefore, the 6 h hydrolysate was selected to determine antioxidant and free radical-scavenging activities of the peptide mixtures.
2.12. Reducing power The reducing power of WGPH was measured according to the method of Oyaizu [18]. Sample (10, 20, 30 and 40 mg) was added to 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 8C for 20 min. Then 2.5 mL of 10% TCA was added to the reaction mixture. A volume of 2.5 mL from each incubated mixture was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride in a test tube. After a 10 min reaction time, the absorbance of the resulting solution was measured at 700 nm. Triplicate tests were conducted for each sample. Increased absorbance of the reaction mixture indicated increased reducing power.
2.13. Determination of ferrous ion-chelating activity Ferrous ion-chelating activity was determined according to the method of Dinis et al. [19]. The hydrolysate samples (3 mL) were added to a solution of
Fig. 1. Hydrolysis curve of defatted wheat germ protein isolates treated with Alcalase 2.4L FG. Reaction conditions: pH, 8.0; temperature, 50 8C; enzyme– substrate ratio, 0.4 AU/g of protein; substrate concentration, 10% (w/v).
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Table 1 Comparative amino acid profile of WGPH and wheat germ protein isolates (g/ 100 g protein) Amino acid
Protein isolates
WGPH
Asp Glub Ser His Gly Thr Arg Ala Tyr Cys Val Met Phe Ile Leu Lys Pro Trp
8.88 15.32 4.86 3.13 6.18 4.12 9.47 5.80 3.24 0.56 6.65 2.11 5.08 4.52 7.81 7.07 4.63 n.dc
8.27 14.71 4.45 2.48 8.67 4.17 8.71 6.98 3.08 0.53 6.09 2.16 4.53 4.22 7.05 7.61 5.96 n.d c
Total hydrophobic amino acidsd
42.78
45.67
a
Fig. 2. The molecular weight distribution profiles of WGPH. GPC-HPLC conditions: column, TSK G2000 SWXL column (7.8 i.d. 300 mm, Tosoh); sample, 100 mg/50 mL; mobile phase, 45% acetonitrile/water/trifluoroacetic acid (45/55/0.1, v/v/v); flow rate, 0.5 mL/min; detection, absorbance at 220 nm. The arrows indicate the elution times of molecular mass markers.
a
3.2. Antioxidant activities of WGPH
b c d
The antioxidative activities of WGPH were measured in linoleic acid emulsion system and compared with those of atocopherol and BHT. As shown in Fig. 3, the autoxidation of linoleic acid was effectively inhibited by the addition of WGPH. The antioxidative activity of WGPH was close to that of a-tocopherol, while it was much lower than that of BHT. Therefore, the result indicated that WGPH seemed to contain some antioxidative peptides and amino acids. The amino acid compositions of wheat germ protein isolates and WGPH were analyzed and shown in Table 1. Although Glu, Arg and Asp were the major constituent amino acids of both protein isolates and WGPH, the amino acid composition of WGPH was different from that of protein isolates. The contents of Gly, Lys, Ala and Pro in WGPH were much higher than those of protein isolates. In addition, the total content of hydrophobic amino acids of WGPH was
Aspartic acid + asparagine. Glutamic acid + glutamine. Not determined. Csontaining Gly, Ala, Val, Leu, Pro, Met, Phe, Trp and Ile.
higher than that of protein isolates. For protein hydrolysates and peptides, an increase in hydrophobicity will increase their solubility in lipid and therefore enhances their antioxidative activity [20,21]. Six peptides from digests of a soybean protein were reported to exhibit high antioxidative activities against lipid peroxidation [22]. These peptides were composed of 5– 16 amino acid residues, including hydrophobic amino acids, Val or Leu, at the N-terminal positions, and Pro, His, or Tyr in the sequences. Chen et al. [23] reported that the N-terminal histidine could contribute higher antioxidative activity to the peptides. In addition, several amino acids, such as Tyr, Met, His, Lys, and Trp, are generally accepted as antioxidants in spite of their pro-oxidative effects in some cases [24]. These information seem to be important in explaining how the hydrolysates possess their antioxidative activity. 3.3. Scavenging effect on DPPH free radical
Fig. 3. The antioxidant activity of WGPH. WGPH was incubated in a linoleic acid emulsion system for 6 days as described in Section 2. The degree of linoleic acid oxidation was measured by the ferric thiocyanate method at every 24 h interval. Butylated hydroxytoluene and a-tocopherol were used as positive controls.
The DPPH free radicals, which are stable in ethanol, show maximum absorbance at 517 nm. When DPPH radicals encounter a proton-donating substance such as an antioxidant, the radicals would be scavenged and the absorbance is reduced. Thus, the DPPH radicals were widely used to investigate the scavenging activity of some natural compounds. Fig. 4 shows the results of scavenging DPPH radical ability of WGPH at various concentrations. WGPH showed dose-dependent DPPH radicals scavenging activity. The EC50 (meaning the concentration that causes a decrease in the initial DPPH concentration by 50%) is a parameter widely used to measure the antiradical efficiency. The lower the EC50, the higher the free radicalscavenging ability. The EC50 value of WGPH for scavenging
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Fig. 4. Scavenging effect on DPPH free radical of different concentrations of WGPH. Butylated hydroxytoluene (BHT) and ascorbic acid were used as positive controls to compare the scavenging effect of WGPH. Regression equation was obtained from linear regression of the concentrations of WGPH and DPPH radical-scavenging effects. Each value is expressed as mean S.D. (n = 3).
activity against DPPH was calculated from the regression equation to be 1.30 mg/mL. The result showed that WGPH is a free radical inhibitor, as well as a primary antioxidant that reacts with free radicals, which may limit the occurrence of free radical damage in human body. 3.4. Superoxide radical-scavenging activity Superoxide radical is known to be very harmful to cellular components as a precursor of more reactive oxidative species, such as single oxygen and hydroxyl radicals [25]. Furthermore, superoxide radical is considered to play an important role in the peroxidation of lipids [26]. Therefore, studying the scavenging effects of WGPH on superoxide radicals is one of the most important ways of clarifying the mechanism of antioxidant activity. The superoxide radical-scavenging activity of WGPH was determined using the cellular xanthine/xanthine oxidase system as a superoxide source. The result is presented in Fig. 5. The scavenging activity of WGPH on superoxide radicals increased with increasing concentrations. The EC50 value of WGPH for superoxide radicals was 0.40 mg/mL. These results indicated that WGPH had a notable effect on scavenging of superoxide radicals. 3.5. Hydroxyl radical-scavenging activity
Fig. 6. Hydroxyl radical-scavenging activities of different concentrations of WGPH by the deoxyribose method. Butylated hydroxytoluene (BHT) was used as positive control. Regression equation was obtained from linear regression of the concentrations of WGPH and hydroxyl radical-scavenging effects. Each value is expressed as mean S.D. (n = 3).
damage and hence human diseases [27]. Among the oxygen radicals specifically, the hydroxyl radical is the most reactive and severely damages adjacent biomolecules such as all proteins, DNA, PUFA, nucleic acid, and almost any biological molecule it touches. This damage causes aging, cancer and several diseases [28]. Therefore, the removal of hydroxyl radical is probably one of the most effective defenses of a living body against various diseases. The scavenging effect against hydroxyl radicals was investigated by using the 2-deoxyribose oxidation method. Fig. 6 shows the hydroxyl radical-scavenging effects of WGPH. The scavenging effect of WGPH on hydroxyl radicals was concentration-dependent. WGPH at the final concentration of 0.16 mg/mL exhibited 67.8% scavenging effect on hydroxyl radical, and the EC50 value was 0.12 mg/mL. According to the present findings, WGPH might be used to provide a good hydroxyl radical scavenger for humans and foods. 3.6. Reducing power For the measurements of the reductive ability, we investigated the Fe3+–Fe2+ transformation in the presence of WGPH using the method of Oyaizu [18]. The reducing power of WGPH was excellent and increased steadily with increasing amount of sample (Fig. 7). At 40 mg, the reducing power was higher than 1.45. The reducing power of bioactive compounds
The reactive oxygen radicals are unstable, and react readily with other groups or substances in the body, resulting in cell
Fig. 5. Superoxide radical-scavenging activities of different concentrations of WGPH in the cellular xanthine/xanthine oxidase system by the NBT method. Each value is expressed as mean S.D. (n = 3).
Fig. 7. Reducing power of different amounts of WGPH using spectrophotometric detection of the Fe3+–Fe2+ transformations. Regression equation was obtained from linear regression of the amounts of WGPH and the absorbances at 700 nm. Each value is expressed as mean S.D. (n = 3).
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References
Fig. 8. Ferrous ion-chelating activities of different concentrations of WGPH. EDTA was used as positive control. Each value is expressed as mean S.D. (n = 3).
had been reported to be associated with their antioxidant activity [29,30]. Thus, a relation should be located between reducing power and the antioxidant effect. Our data on the reducing power of WGPH suggested that it was likely to contribute significantly towards the observed antioxidant effect. 3.7. Ferrous ion-chelating activity The chelating of ferrous ions by WGPH was estimated by the method of Dinis et al. [19]. Ferrozine can quantitatively form complexes with Fe2+ ion. In the presence of chelating agents, the complex formation is disrupted resulting to a decrease in the red colour of the complex. Measurement of colour reduction therefore makes possible the estimation of the metal chelating activity of the coexisting chelator [31]. In this assay, WGPH as well as EDTA interfered with the formation of ferrous and ferrozine complex, thus suggesting that it has a chelating activity and captures ferrous ion before ferrozine. EDTA is a known metal ion chelator; and therefore the chelating effect of WGPH was compared with it. The ferrous ion-chelating effects of WGPH and EDTA are shown in Fig. 8. WGPH showed a strong chelating activity on Fe2+ ion. It displayed 89.0% chelating effect on Fe2+ ion at a concentration of 1000 mg/mL. However, the chelating ability was slightly lower than that of EDTA. This indicated that WGPH had an effective capacity for iron binding, suggesting that its action as an antioxidant may be related to its iron binding capacity. 4. Conclusion The enzymatic hydrolysis of wheat germ protein through the action of Alcalase 2.4L FG provided a high proportion of peptides from 1500 Da to free amino acids. Freeze-dried WGPH showed a relatively higher antioxidant activity and free radical-scavenging activities. At present, consumer demand for natural functional foods, has been increasing, and therefore WGPH will be used as a functional food ingredient in pharmaceutical and food industries in the future. However, further detailed studies on WGPH and its peptide fractions with regard to antioxidant activity in vivo and the different antioxidant mechanisms are needed.
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