Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods

Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health.


Introduction
Sericin protein from the cocoon of silkworm (Bombyx mori) is regarded as the resistant protein against digestion together with buckwheat and soybean proteins (1)(2)(3). Sericin is a type of protein created by silkworms in the production of silk. Silk emitted by the silkworm consists of two main proteins, sericin and fibroin, with fibroin being the structural center of the silk, and sericin being the sticky material surrounding it. Notably, during the production of silk cloth, the sericin protein is discarded.
In a previous study, we reported the dyeing property of silkworm cocoon (4,5). Lipid oxidation on vegetable oil and frying oil using the indicators peroxide value (POV), carbonyl value (COV) and anisidin value (AnV) has been previously identified (6,7). Additionally, recent studies have focused on the antioxidative activity of seafood using oxygen radical absorbance capacity (ORAC), hydroxyl radical averting capacity (HORAC) and electron spin resonance (ESR) methods (8,9).
By contrast, Dash et al showed that sericin protein from silkworm inhibited UVB-induced apoptosis in human skin keratinocytes (10), and that the antioxidant potential of the protein against hydrogen peroxide-induced oxidative stress in skin fibroblasts using the indicators of catalase, lactate dehydrogenase and malondialdehyde activities (11). Zhaorigetu et al demonstrated that sericin protein exerts a suppressant effect on skin cancer (12) and colon cancer (13), while Li et al demonstrated a protective effect of sericin and sericin peptide against alcohol-induced liver and gastric injuries in mice (14,15). Aramwit et al (16) and Ohnishi et al (17) identified a cell-protective effect of sericin on collagen and nitric oxide, and cryopreserved rat islets, respectively. Additionally, Manosroi et al (18) and Jin-Bo et al (19) identified the antioxidant and tyrosinase inhibition activity of sericin protein extracted from silkworm.
Efforts have been made to determine whether the utilization of sericin proteins extracted from cocoon of silkworm (Bombyx mori) may be beneficial for human consumption.
One of the physico-chemical properties of sericin is its antioxidant activity (20). Cocoon shells vary in color, i.e., white, green, yellow and red. The color components coexist and accumulate in the sericin layer of cocoons. Among different colored cocoons, yellow-green cocoon shells contain various flavonol compounds with antioxidant activity (21). In this study, we investigated the antioxidant activities of different types of sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, using the 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, ORAC and ESR methods. Furthermore, we examined whether or not the antioxidant activity of bread was elevated by the addition of sericin protein.

Materials and methods
Samples. White sericin and yellow-green sericin powder was used. Sericin powder was also added to bread, while additive-free bread served as the control. White sericin powder was purchased from Kashiro Sangyo Co. Ltd. (Shiga, Japan) and yellow-green sericin powder was purchased from Shiono-ya (Kyoto, Japan). Bread was used as an example of processed food to which sericin powder was added.
Sample preparation. To measure the antioxidant activity, each sample (5 g) was homogenized with 5 ml water using a mortar for 2 min. Then, 15 ml of water was added and the mixture was homogenized for 1 min. The sample solutions were centrifuged at 15,000 x g for 15 min. The supernatant was removed and the sample was frozen at -30ºC as the stock sample solutions.

Measurement of DPPH radical scavenging activity.
Measurement of DPPH radical scavenging activity for all fractions was performed using the method by Yamaguchi et al (22). Briefly, an aliquot of antioxidant solution (200 µl) was mixed with 100 mM Tris-HCl buffer (pH 7.4, 800 µl) and added to 1 ml of 500 µM DPPH in EtOH. The mixture was agitated vigorously, left to stand for 20 min at room temperature in the dark, and then subjected to reverse-phase high performance liquid chromatography (HPLC) analysis. The HPLC equipment consisted of a Shimadzu LC-6A pump, a Rheodyne injector fitted with a 20 µl loop and a Shimadzu SPD-6AV UV-VIS detector set at 517 nm (0.064 AUFS). Analyses were conducted in a TSKgel Octyl-80Ts column (4.6x250 mm, Tosoh, Tokyo, Japan) at room temperature with a mobile phase of MeOH/H 2 O (7:3, v/v) at a flow rate of 0.8 ml/min. The DPPH radical-scavenging activity was evaluated from the difference in peak area decrease of the DPPH radical detected at 517 nm between a blank and a sample. Trolox was used as a control standard. The data were expressed as mmol of Trolox equivalent per 100 ml of each sericin sample.
Chemiluminescence experiment procedure. The chemiluminescence method has been described in detail previously (23). Briefly, the AAPH (40 mM) reagent was dissolved in 100 mM phosphate buffer (pH 7.0). The sericin sample solutions were also diluted to the appropriate concentration using the same buffer. The AAPH solution was heated at 37˚C for 2 min to generate peroxyl radicals. The AAPH solution (0.2 ml) was then mixed with 0.2 ml phosphate buffer as the control or with 0.2 ml diluted sericin sample, and the solution mixture was then heated at 37˚C for 2 min. For exposure to heating, a water bath (Thermo Max TM-1, AS ONE Corporation, Osaka, Japan) was used. The temperature was maintained within ±3˚C. Immediately after heating, 0.2 ml luminol solution was added to the mixture for chemiluminescence measurement. For the luminol solution, luminol (0.113 mM) and cytochrome c (0.004 mM) were dissolved in a mixture of 100 mM sodium tetraborate buffer (pH 9.28), water and methanol (volume ratio, 9:1:30). Final concentrations of AAPH, luminol and cytochrome c were 13.333, 0.038 and 0.001 mM, respectively. Chemiluminescence intensity was measured using a photon counter Lumitester C-110 (Kikkoman Co., Tokyo, Japan), where one relative light unit (RLU) represented ~43 photons/sec.
Calculation of the IC 50 of peroxyl radical scavenging. As an indicator of antioxidative capacity, the inhibition of chemiluminescence intensity was measured by the change of RLU value. The lower the RLU value the more inhibition of chemiluminescence intensity. The value of IC 50 was defined as the concentration of the sericin sample that reduced the  (9 The standard regression line was obtained by plotting the trolox concentrations against the net AUC trolox of each concentration: Net AUC trolox = AUC trolox -AUC control , and Net AUC sample = AUC sample -AUC control ; where AUC trolox is the AUC in the presence of trolox; AUC control is the AUC with blank control and AUC sample is the AUC with sample buffer. The horizontal axis is the net AUC trolox and the vertical axis is the concentration of trolox. The equation Y = ax + b was derived from the above data and the values for a and b were obtained. The Electron spin resonance analysis. ESR was conducted as previously described (8). Briefly, hydroxyl radical generation was first examined by the DMPO method and iron (II) sulfate with or without the fish sauce samples. The addition of 8.8 mM H 2 O 2 (50 µl) to the reaction mixture (320 µl) was used to initiate Fenton's reaction as depicted in the chemical equation: Fe 2+ + H 2 O 2 → Fe 3+ + •OH + OH -. After l min of hydroxyl radical generation, spin adduct DMPO-OH• was measured using the ESR spectrometer (JES-FR30: JEOL Ltd., Tokyo, Japan). ESR measurement conditions were: output, 4 mW (9.4 GHz); magnetic field, 342.790±5 mT; modulation amplitude, 0.079 mT; time constant, 0.1 sec; sweeping time, l min; amplification ratio, 32-125.
Calculation of the IC 50 of hydroxyl radical scavenging. IC 50 values were defined as the concentration of each sericin sample that reduced the control peak height ratio of ESR (generation of hydroxyl radical) by half. The antioxidative value was calculated using the formula: (log Io/I) x 100, where Io is the ESR peak height ratio as the control, and I is the ESR peak height ratio as a sample. Thus, the IC 50 value was the concentration of samples at Io/I = 1/2, calculated from the antioxidant results of ESR obtained in the experiments.
Statistical analysis. The Fisher's exact test, Student's t-test and correlation analysis were performed using Microsoft excel.

Results and Discussion
Measurement of radical scavenging activity by the DPPH-HPLC method. Measured results are shown in Table Ⅱ. DPPH is a stable, artificial-free radical. Yamaguchi et al reported that the DPPH radical scavenging activities of onion, broccoli, and burdock were 104, 890, and 3685 µmol Trolox eq./100 g, respectively (24,25). When compared with these values, yellow-green and white sericin proteins were 23.7 and 18.2 mmol Trolox eq./100 g, respectively, indicating high DPPH radical scavenging activities. The value of yellow-green sericin was significantly higher than that of white sericin. Higher DPPH radical scavenging activity was presumably caused by the additional effect of flavonoid pigment contained in yellow-green sericin. Thus, sericin proteins may demonstrate a high DPPH radical scavenging activity.
Measurement of peroxyl radical scavenging activity by chemiluminescence method. Measured results are shown in Fig. 1. The yellow-green and white sericin proteins indicated high peroxyl radical scavenging activities. Nagao et al reported that the IC 50 value of the peroxyl radical scavenging activity was 0.1% in kale, which is known to exhibit a high antioxidative property (water-extracted sample) (26). Specifically, the antioxidant activity of yellow-green sericin protein < 0.01% of the IC 50 value was significantly higher than that of the white sericin protein of ~0.05% of IC 50 value (p<0.01). In the same manner as the DPPH radical scavenging activity, it is considered that the    higher peroxyl radical scavenging activity was caused by additional effect of flavonoid pigment contained in yellow-green sericin. In addition, bread with sericin powder had significantly high peroxyl radical scavenging activity when compared with bread without sericin powder (p<0.01). Improvement of peroxyl radical scavenging activity was observed by the addition of sericin (~4% of bread powder).
Measurement of peroxyl radical eliminating activity by the ORAC method. As shown in Table Ⅲ, the peroxyl radical eliminating activity was measured by the ORAC method. It has been reported by Yamaguchi et al (25) that peroxyl radical eliminating activities of burdock, ginger, and komatsuna (Japanese mustard spinach) were 7.5, 4.8, and 12.3 mmol Trolox eq./100 g, respectively, and the activity of kamaboko was also reported by Harada et al (9) as 0.166 mmol Trolox eq./100 g. Particularly, the value of yellow-green sericin protein (29.3 mmol Trolox eq./100 g) was significantly higher than that of the white sericin protein (10.0 mmol Trolox eq./100 g) (p<0.05). Similar to the DPPH and peroxyl radical scavenging activities, higher peroxyl radical eliminating activity was presumably the result of flavonoid pigment contained in yellow-green sericin protein.
Measurement of hydroxyl radical scavenging activity by the ESR method. As shown in Fig. 2, the hydroxyl radical scavenging activity was measured by the ESR method. The yellow-green and white sericin proteins indicated high antioxidant activity wherein the activity of white sericin protein was higher than that of yellow-green sericin protein although there was no significant difference. By using all the above-mentioned methods, yellow-green sericin indicated higher values. Based on these results, it is assumed that the antioxidant capacity of flavonoid pigment may not be involved in the elimination of hydroxyl radicals. Additionally, bread with sericin powder had significantly high hydroxyl radical scavenging activity when compared with bread without sericin. As for the hydroxyl radical scavenging activity, the improvement of hydroxyl radical scavenging activity was observed by the addition of sericin (~4 % of bread powder) (Fig. 2).
Correlation between DPPH radical scavenging activity and ORAC value. Based on the values obtained from the four types of samples, the correlation coefficient between the DPPH radical scavenging activity and ORAC value was calculated (Fig. 3). As shown in Fig. 3, a positive correlation of R 2 =0.85 was observed. Conflicting reports have concluded that DPPH and ORAC methods have no correlation (27) or that the methods are correlated (28). By contrast, a positive correlation was observed in the case of sericin proteins and bread to which sericin powder was added.
Antioxidant property of sericin proteins. Antioxidative mechanisms of sericin proteins, as well as the scavenging and eliminating components remain to be elucidated. Kato et al hypothesized that the scavenging function may be provided by the chelating effect of hydroxyl groups of hydroxyamino acids (serine and threonine) that are abundantly contained in sericin (20). Sericin has clearly demonstrated antioxidant capacities against multiple radicals through the measurement of radical scavenging (eliminating) activities by four different methods in this study. Thus, sericin proteins may be efficiently utilized as beneficial for human consumption.