Gelatin and Antioxidant Peptides from Gelatin Hydrolysate of Skipjack Tuna (Katsuwonus pelamis) Scales: Preparation, Identification and Activity Evaluation

For full use of fish by-products, scale gelatin (TG) and antioxidant peptides (APs) of skipjack tuna (Katsuwonus pelamis) were prepared, and their properties were characterized using an amino acid analyzer, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Fourier transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometers (ESI-MS), and radical scavenging assays. The results indicate that TG with a yield of 3.46 ± 0.27% contained Gly (327.9 ± 5.2 residues/1000 residues) as the major amino acid and its imino acid content was 196.1 residues/1000 residues. The structure of TG was more unstable than that of type I collagen from scales of skipjack tuna (TC) and TG was more suitable for preparation of hydrolysate by protease than mammalian gelatins. Therefore, TG was separately hydrolyzed under five proteases (pepsin, papain, trypsin, neutrase, and alcalase) and ten APs (TGP1–TGP10) were isolated from the alcalase-hydrolysate. Among them, TGP5, TGP7, and TGP9 with high antioxidant activity were identified as His-Gly-Pro-Hyp-Gly-Glu (TGP5), Asp-Gly-Pro-Lys-Gly-His (TGP7) and Met-Leu-Gly-Pro-Phe-Gly-Pro-Ser (TGP9), respectively. Furthermore, TGP5, TGP7, and TGP9 exhibited a high radical scavenging capability on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (EC50 values of 1.34, 0.54, and 0.67 mg/mL, respectively), hydroxyl radical (EC50 values of 1.03, 0.41, and 0.74 mg/mL, respectively), and superoxide anion radical (EC50 values of 1.19, 0.71, and 1.59 mg/mL, respectively). These results suggest that three APs (TGP5, TGP7, and TGP9), especially TGP7, have a strong antioxidant activity and could act as potential antioxidant ingredients applied in functional products.


Introduction
Gelatin is a denatured form of collagen and traditionally extracted by processing by-products of land mammals, such as beef bones, bovine hides, and pig skins [1][2][3]. According to the acid and alkali preparation processes, the produced gelatins were divided into type A and type B, with a and identify the APs from the gelatin hydrolysate, and evaluate the radical scavenging activities of isolated APs.  Table 1, the yield of TG was 3.46 ± 0.27% (on a dry scale weight basis), which was significantly lower than those of gelatins from skins (11.3 ± 0.03%) [24] and bones (6.37 ± 0.64%) [4] of skipjack tuna. The protein content of TG was 94.08 ± 4.52 g/100 g, which was significantly higher than the contents of moisture (3.78 ± 0.39%), fat (0.53 ± 0.22%), and ash (1.05 ± 0.16) in the TG. In addition, the protein content of TG was significantly higher than those of gelatins from the skins (88.4 ± 0.12 g/100 g) and bones (90.14 ± 3.98 g/100 g) of skipjack tuna [25]. All values are mean ± standard deviation (SD) (n = 3).

Amino Acid Composition of TG
As shown in Table 2, the amino acid pattern of gelatin (TG) from the scales of skipjack tuna was similar to that of type I collagen from scales of skipjack tuna (TC). Glycine (Gly) was the highest content of amino acid of TG and TC with contents of 327.9 ± 5.2 and 330.6 ± 4.6 residues/1000 residues, respectively. The reason is that about 50-60% of α-chains in collagen are composed of typical tripeptide repetitions (Gly-X-Y) [26,27]. In addition, TG and TC were rich in alanine (Ala), proline (Pro), and hydroxyproline (Hyp) with a descending order. The amino acid pattern of gelatin (TG) was similar to those of gelatins from bovine heart [28] and skin [5], tuna bones and skin [4,24], and salmon, rohu and shark skins [24,29].
The amount of imino acids (Pro and Hyp) is closely related to the stability of the gelatins and collagens because the pyrrolidine rings of Pro and Hyp can assist in maintaining the stability of the triple helical structure [4,30]. In addition, the hydroxyl group in Hyp can form intermolecular hydrogen bonds to reinforce the triple-stranded helix of gelatins [4,24]. Table 2 shows that the content of imino acids of TG was 196.1 residues/1000 residues, which is slightly higher than those of gelatins from dover sole skins (173-183 residues/1000 residues) [31], tuna bones (177.3 residues/1000 residues) [4], and bigeye snapper skins (186-187 residues/1000 residues) [32], but significantly lower than that of bovine gelatin (219.0 residues/1000 residues) [33]. The data indicate that the helices of TG were more unstable and easier to hydrolysis than bovine gelatin.  All values are mean ± standard deviation (SD) (n = 3). ND = not detected.

Electrophoretic Pattern of TG
The MW distribution and subunit compositions can significantly affect the properties of gelatins and collagens. Figure 1 presents the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of TG and TC. The image indicates that TG had similar protein patterns to those of TC. They were composed of two α-chains (α1 and α2 chains with the band intensity ratio of 2:1). The pattern of TG ([α1] 2 α2) was similar to those of gelatins from seafood by-products, such as the bones of skipjack tuna [4] and the skins of shark, rohu, tuna [24] and carp (Cyprinus carpio) [30]. In addition, β-chain (α-chain dimer) were also found in the protein patterns of TG and TC, but the band intensities of γ chain (α-chain trimer) of TC were significantly stronger than those of TG. A series of peptide fragments with MW below 100 kDa was noticeable in the protein patterns of TG. These results indicate that partial triple helical structure and peptide bonds of TG were degraded during the heating extraction process [4,34]. All values are mean ± standard deviation (SD) (n = 3). ND = not detected.

Electrophoretic Pattern of TG
The MW distribution and subunit compositions can significantly affect the properties of gelatins and collagens. Figure 1 presents the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of TG and TC. The image indicates that TG had similar protein patterns to those of TC. They were composed of two α-chains (α1 and α2 chains with the band intensity ratio of 2:1). The pattern of TG ([α1]2α2) was similar to those of gelatins from seafood by-products, such as the bones of skipjack tuna [4] and the skins of shark, rohu, tuna [24] and carp (Cyprinus carpio) [30]. In addition, β-chain (α-chain dimer) were also found in the protein patterns of TG and TC, but the band intensities of γ chain (α-chain trimer) of TC were significantly stronger than those of TG. A series of peptide fragments with MW below 100 kDa was noticeable in the protein patterns of TG. These results indicate that partial triple helical structure and peptide bonds of TG were degraded during the heating extraction process [4,34]. The FTIR spectral profiles of TG and TC are shown in Figure 2 and the wavenumber of five major peaks (Amide A, B and I-III) are presented in Table 3. The spectrum of TG was similar to those of TC and gelatins from bones of skipjack tuna [4] and golden carp skins [35]. The FTIR spectral profiles of TG and TC are shown in Figure 2 and the wavenumber of five major peaks (Amide A, B and I-III) are presented in Table 3. The spectrum of TG was similar to those of TC and gelatins from bones of skipjack tuna [4] and golden carp skins [35].  Amide I, II, and III bands are caused by C=O stretching, N-H bending, and C-H stretching, respectively. They are bound up with the triple helical structures of gelatins [4,35]. A lower wavenumber amide I bands (1600-1700 cm −1 ) indicates a reduction of the gelatin molecular order [35,36]. The wavenumber of TG (1667 cm −1 ) was lower than that of TC (1689 cm −1 ), which suggests that a fraction of telopeptides of TG were hydrolyzed during the heating preparation process. A low wavenumber of the amide II band suggests that gelatin has a higher structure order because it forms more hydrogen bonding by N-H groups [26,36]. The wavenumber of TG was found to be 1536 cm −1 , which was higher than that of TC (1484 cm −1 ), which suggests that TG has less hydrogen bonding than TC. The wavenumber of amide III band is related to the triple helix structure of gelatin. In Figure 2, the amide III bands of TG and TC are located at wavenumbers of 1215 cm −1 and 1209 cm −1 , respectively. The data suggests that more α-helix structure was converting to random coils upon heating extracting process. The wavenumber of free N-H groups (3400-3440 cm −1 ) was removed to a lower frequency, indicating the N-H group of the peptide involved in hydrogen bonding [4,35]. Figure 2 shows that the amide A wavenumbers of TC (3325 cm −1 ) and TG (3351 cm −1 ) suggest that the degree of hydrogen bonding in TC was more than that of TG. The amide B band is related to the asymmetric stretch vibrations of -NH + 3 and =C-H, and the high wavenumber of amide B band indicates an increase in free NH-NH + 3 groups. The wavenumbers of the amide B of TG (2939 cm −1 ) were lower than that of TC (2977 cm −1 ). The data suggests that TG has freer -NH + 3 groups than TC because it formed fewer hydrogen bonds compared with TC.
Based on the results of amino acid composition, SDS-PAGE patterns, and FTIR spectra of TG, we found that the structure of TG was more unstable than TC. Therefore, TG should be suitable to prepare active peptides because of its unstable structure.

Preparation of TG Hydrolysate
As shown in Table 4, TG was separately hydrolyzed under five kinds of proteases, and the alcalase-hydrolysate (TGH) showed the highest degree of hydrolysis (DH) (25.35 ± 1.68%) and  Amide I, II, and III bands are caused by C=O stretching, N-H bending, and C-H stretching, respectively. They are bound up with the triple helical structures of gelatins [4,35]. A lower wavenumber amide I bands (1600-1700 cm −1 ) indicates a reduction of the gelatin molecular order [35,36]. The wavenumber of TG (1667 cm −1 ) was lower than that of TC (1689 cm −1 ), which suggests that a fraction of telopeptides of TG were hydrolyzed during the heating preparation process. A low wavenumber of the amide II band suggests that gelatin has a higher structure order because it forms more hydrogen bonding by N-H groups [26,36]. The wavenumber of TG was found to be 1536 cm −1 , which was higher than that of TC (1484 cm −1 ), which suggests that TG has less hydrogen bonding than TC. The wavenumber of amide III band is related to the triple helix structure of gelatin. In Figure 2, the amide III bands of TG and TC are located at wavenumbers of 1215 cm −1 and 1209 cm −1 , respectively. The data suggests that more α-helix structure was converting to random coils upon heating extracting process. The wavenumber of free N-H groups (3400-3440 cm −1 ) was removed to a lower frequency, indicating the N-H group of the peptide involved in hydrogen bonding [4,35]. Figure 2 shows that the amide A wavenumbers of TC (3325 cm −1 ) and TG (3351 cm −1 ) suggest that the degree of hydrogen bonding in TC was more than that of TG. The amide B band is related to the asymmetric stretch vibrations of -NH 3 + and =C-H, and the high wavenumber of amide B band indicates an increase in free -NH 3 + groups. The wavenumbers of the amide B of TG (2939 cm −1 ) were lower than that of TC (2977 cm −1 ). The data suggests that TG has freer -NH 3 + groups than TC because it formed fewer hydrogen bonds compared with TC.
Based on the results of amino acid composition, SDS-PAGE patterns, and FTIR spectra of TG, we found that the structure of TG was more unstable than TC. Therefore, TG should be suitable to prepare active peptides because of its unstable structure.

Preparation of TG Hydrolysate
As shown in Table 4, TG was separately hydrolyzed under five kinds of proteases, and the alcalase-hydrolysate (TGH) showed the highest degree of hydrolysis (DH) (25.35 ± 1.68%) and HO· scavenging activity (29.46 ± 1.37%) at a concentration of 5.0 mg/mL among five kinds of hydrolysates. The specificity of protease serving for the hydrolysis process is the most important factor of the preparation of biopeptides [14]. As shown in Table 4, the protein hydrolysates revealed significantly different DH and antioxidant activities, mainly due to the various spectra of hydrolysate specificities, such as chain length, amino acid sequence, and spatial structure [14,37]. As an endo-protease, alcalase showed a strong proteolytic activity and was wildly used in hydrolyzing seafoods and their byproducts, such as Antartic krill [38], tuna backbone, black muscle and heads [23,39,40], sardinelle heads and viscera [41], and croceine croaker muscle [42]. In this experiment, TGH exhibited the highest radical scavenging activity and was chosen for the following separation process of APs. The data are presented as the mean ± SD (n = 3). a-c Values with same letters in each column indicate no significant difference (p > 0.05).

Purification of APs from TGH
Using 3 and 5 kDa ultrafiltration membranes, three fractions, including TGH-I (<3 kDa), TGH-II (3-5 kDa), and TGH-III (>5 kDa), were prepared from TGH and their HO· scavenging activity are shown in Figure 3. The HO· scavenging activity of TGH-I was 38.19 ± 2.35% at a concentration of 5.0 mg/mL, which was significantly higher than those of TGH (29.46 ± 1.37 mg/mL), TGH-II (27.42 ± 1.46%), and TGH-III (19.36 ± 0.97%) (p <0.05). The lowest MW fraction (TGH-I) having the strongest activity is consistent with previous reports that the antioxidant capability of hydrolysates was inversely related to their average MW because they were more easily contacted with free radicals [2,43]. Then, TGH-I was further purified using chromatographic methods. factor of the preparation of biopeptides [14]. As shown in Table 4, the protein hydrolysates revealed significantly different DH and antioxidant activities, mainly due to the various spectra of hydrolysate specificities, such as chain length, amino acid sequence, and spatial structure [14,37].
As an endo-protease, alcalase showed a strong proteolytic activity and was wildly used in hydrolyzing seafoods and their byproducts, such as Antartic krill [38], tuna backbone, black muscle and heads [23,39,40], sardinelle heads and viscera [41], and croceine croaker muscle [42]. In this experiment, TGH exhibited the highest radical scavenging activity and was chosen for the following separation process of APs. The data are presented as the mean ± SD (n = 3). a-c Values with same letters in each column indicate no significant difference (p > 0.05).
As shown in Figure 5A, AC-III was further divided into three fractions (GC-І to GC-ІII) using a Sephadex G-25 column. The HO· radical scavenging activity of GC-IIІ was 65.79 ± 4.21% at a concentration of 5.0 mg/mL, which was significantly stronger than those of TGH (29.46 ± 1.37%), TGH-I (38.19 ± 2.35%), AC-III (47.72 ± 3.11%), GC-I (32.46 ± 2.65%), and GC-II (40.78 ± 3.04%) (p <0.05) ( Figure 4B). Then, GC-ІII was selected for the following reversed-phase high-performance liquid chromatography (RP-HPLC) separation process. As shown in Figure 6, GC-ІII was finally purified using the RP-HPLC system with a Zorbax C-18 column, and the eluted peptides were gathered separately on their chromatographic peaks. At last, ten peptides with retention times of 5.006 min (TGP1), 5.067 min (TGP2), 10.535 min (TGP3), As shown in Figure 6, GC-III was finally purified using the RP-HPLC system with a Zorbax C-18 column, and the eluted peptides were gathered separately on their chromatographic peaks. At last, ten peptides with retention times of 5.   Figure 7 shows the HO· scavenging activity of ten isolated APs (TGP1 to TGP10). At a concentration of 5.0 mg/mL, the HO· scavenging activities of TGP5, TGP7, and TGP9 were 80.51 ± 3.05%, 85.66 ± 2.68%, and 82.41 ± 2.34%, respectively, which is significantly higher than those of the other seven peptides. Therefore, TGP5, TGP7, and TGP9 were chosen for an amino acid sequence analysis.  Figure 7 shows the HO· scavenging activity of ten isolated APs (TGP1 to TGP10). At a concentration of 5.0 mg/mL, the HO· scavenging activities of TGP5, TGP7, and TGP9 were 80.51 ± 3.05%, 85.66 ± 2.68%, and 82.41 ± 2.34%, respectively, which is significantly higher than those of the other seven peptides. Therefore, TGP5, TGP7, and TGP9 were chosen for an amino acid sequence analysis. chromatography (RP-HPLC) on a Zorbax, SB C-18 column (4.6 mm × 250 mm) from 0 to 30 min. Figure 7 shows the HO· scavenging activity of ten isolated APs (TGP1 to TGP10). At a concentration of 5.0 mg/mL, the HO· scavenging activities of TGP5, TGP7, and TGP9 were 80.51 ± 3.05%, 85.66 ± 2.68%, and 82.41 ± 2.34%, respectively, which is significantly higher than those of the other seven peptides. Therefore, TGP5, TGP7, and TGP9 were chosen for an amino acid sequence analysis. Figure 7. HO· scavenging activity of ten major sub-fractions (TGP1 to TGP10) of GC-ІII at the concentration of 5.0 mg/mL. The data are presented as the mean ± SD (n = 3). a-f The column wise values with same superscripts indicate no significant difference (p > 0.05).

Relationship among Molecular Size, Amino Acid Composition, and Antioxidant Activity
Structural properties offer guidance for speculating the bioactivities of peptides and predicting their potential applications. To date, most reports suggested that MW, hydrophobicity, and the composition and sequence of amino acids were affecting the antioxidant ability of APs [14,50]. The three APs (TGP5, TGP7, and TGP9) from the gelatin hydrolysate of skipjack tuna scales are hexapeptide to octapeptide with MWs ranging from 608.57 Da to 804.92 Da (Table 5), which confirms that TGP5, TGP7, and TGP9 were easily contacted with target radical to play their antioxidant functions.
Due to the hydrophobic properties of polyunsaturated fatty acids (PUFAs), amino acids with a hydrophobic branched chain have a high reactivity to them for helping them avoid the radical damage [47,51,52]. Furthermore, aromatic groups can stabilize radicals through donating protons to terminate the oxidative stress reaction [44,53]. Consequently, hydrophobic/aromatic amino acids, such as His, Pro, Met, Leu, and Phe, are regarded as the most important antioxidant factor of peptides [5,14]. For example, Jin et al. reported that the high antioxidant activities of MCLDSCLL and HPLDSLCL were duo to the amino acid residues of Leu, Met, and His in their sequences [54]. Therefore, His, Pro, and Hyp in the sequence of TGP5, Pro and His in the sequence of TGP7, and Met, Leu, Pro, and Phe in the sequence of TGP9 could significantly increase their antioxidant capacity.
The hydrophobicity of peptides is important for accessibility to hydrophobic targets and enhances the affinity and reactivity of peptide. However, the composition and ratio of hydrophilic amino acids, such as Asp, Glu, Lys, and Gly, are pivotal in peptide antioxidant activity and are especially considered to be associated with antioxidant effects in vivo [55,56]. For example, the side chains of amino and carboxyl groups in amino acids are important for metal ion chelating and HO· scavenging activities of peptides [57][58][59]. Zhang et al. reported that Asp residues in WMFDW and Glu residues in EMGPA played critical roles in the activities of radical scavenging and lipid peroxidation inhibition [4]. Hu et al. reported that basic (Lys) and acidic (2Asp and Glu) amino acid residues in the sequence of NWDMEKIWD took responsibility for its outstanding activity [60]. In addition, Zhang et al. and Yang et al. reported that Gly residues could expediently donate hydrogen atoms to passivate active radicals because they could make the peptide backbone of gelatin and collagen more flexible [4,61]. Therefore, Gly and Glu in the amino acid sequence of TGP5, Asp, Lys, and 2Gly in the amino acid sequence of TGP7, and 2Gly in the amino acid sequence of TGP9 could play critical roles in their radical scavenging activities.

Materials
Scales of skipjack tuna (K. pelamis) were supplied by Ningbo Todayfood Co. Ltd. (Ningbo, China). DEAE-52 cellulose and Sephadex G-25 were purchased from Shanghai Source Poly Biological Technology Co., Ltd (Shanghai, China). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China). DPPH was purchased from Sigma-Aldrich (Shanghai, China) Trading Co., Ltd. (Shanghai, China). Type I collagen (TC) from the scales of skipjack tuna was prepared in our lab.

Preparation of Scale Gelatin (TG) and Gelatin Hydrolysate of Kipjack Tuna
The pretreatment of scales, including removing non-collagenous proteins and minerals, was performed according to a previous method [61]. Then, the gelatin was extracted from pretreated scales according to the method described by Yang et al. with a slight modification [4]. In brief, the extraction process of gelatin was performed in DW at 60 • C for 8 h with a solid-to-liquid ratio of 1:10 (w/v). Finally, the extracting solution was centrifuged at 12,000 g for 15 min to remove the solid residues. The supernatant, named TG, was collected and lyophilized.
The hydrolytic process of TG was performed following the previous methods [56,61]. The TG dispersions (1%, w/v) were hydrolyzed separately using five proteases in the hydrolysis parameters in Table 6. After 4-hour hydrolysis, the hydrolysate was heated at 90 • C for 20 min and centrifuged at 8000× g for 25 min at room temperature. The resulting supernatants were collected, freeze-dried, and kept at −20 • C. The alcalase-hydrolysate was referred to as TGH. Moisture, ash, fat, and protein contents of the scales and gelatin were measured using the methods of AOAC with the method numbers of 950.46B, 920.153, 960.39 (a), and 928.08, respectively [62]. An amino acid analysis was performed according to the methods described by Zhao et al. [36].

SDS-PAGE
Electrophoretic patterns of TG and TC were performed using 4% stacking gel and 7.5% separating gel [26]. The samples (10 µg proteins) were mixed with the sample loading buffer at a ratio of 4:1 (v/v) in the presence of β-ME, then applied to sample wells and electrophoresed at a constant voltage of 100 V. After about 4 h, the gel was fixed with 10% acetic acid and 50% (v/v) methanol for 0.5 h. The gel was stained for 3 h with a Coomassie blue R-250 solution and de-stained with 10% (v/v) acetic acid and 30% (v/v) methanol solution. A high MW marker was used to estimate the MWs of proteins. TC was used as a collagen standard.

FTIR
The infrared spectra (450-4000 cm −1 ) of TG and TC were recorded in KBr disks with a Fourier transform IR spectrophotometer (Nicolet 6700, Thermo Fisher Scientific Inc., Waltham, MA, USA). The mixture with the sample-to-KBr ratio of 1:100 (w/w) was pressed into a disk for spectrum recording.

Isolation of Peptides from TGH
TGH was fractionated with 3 and 5 kDa MWCO membranes and three fractions, including TGH-I (MW <3 kDa), TGH-II (MW 3-5 kDa), and TGH-III (MW >5 kDa) were collected and lyophilized. An amount of 5.0 mL of TGH-I solution (40.0 mg/mL) was injected into a pre-equilibrated column (1.6 cm × 80 cm) of DEAE-52 cellulose with DW, and stepwise eluted with 150 mL DW and NaCl solution (0.1 M, 0.5 M, and 1.0 M, respectively) at a flow rate of 1.0 mL/min, respectively. Each eluate (5.0 mL) was monitored at 214 nm. Finally, four fractions (AC-I to AC-IV) were collected and lyophilized on their chromatographic peaks. An amount of 5.0 mL of AC-III solution (20.0 mg/mL) was separated on a column (2.6 cm × 160 cm) of Sephadex G-25 and eluted with DW at a flow rate of 0.6 mL/min. Each eluate (3.0 mL) was collected and monitored at 214 nm, and three subfractions (GC-I, GC-II, and GC-III) were collected and lyophilized. GC-III was purified using RP-HPLC with a Zorbax, SB C-18 column (4.6 × 250 mm, 5 µm) on an Agilent 1260 (Santa Rosa, CA, USA). The sample was eluated with a linear gradient of ACN (0%-50% in 0-35 min) in 0.1% TFA at a flow rate of 0.8 mL/min. Ten APs (TGP1 to TGP10) were isolated at an absorbance of 214 nm and lyophilized.

Amino Acid Sequence and MW Analysis
The amino acid sequences of three APs (TGP5, TGP7, and TGP9) were measured on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc, Foster City, CA, USA).
The MWs of three APs (TGP5, TGP7, and TGP9) were determined using a Q-TOF mass spectrometer (Micromass, Waters, Milford, MA, USA) coupled with an electrospray ionization (ESI) source. Ionization was carried out in positive mode with a capillary voltage of 3500 V. Nitrogen was maintained at 40 psi for nebulization and 9 L/min at 350 • C for evaporation temperature. Data were collected in centroid mode from 100 to 2000 m/z.

Antioxidant Activity
The DPPH·, HO·, and O − 2 · scavenging assays of three APs (TGP5, TGP7, and TGP9) were determined by the previous method [56], and the half elimination ratio (EC 50 ) was defined as the concentration where a sample caused a 50% decrease of the initial radical concentration.

DPPH· Scavenging Activity
An amount of 2.0 mL of samples consisting of distilled water and different concentrations of the analytes were placed in cuvettes, and 500 µL of an ethanolic solution of DPPH (0.02%) and 1.0 mL of ethanol were added. A control sample containing the DPPH solution without the sample was also prepared. In the blank, the DPPH solution was substituted with ethanol. The DPPH· scavenging activity was calculated using the following formula: where A s is the absorbance rate of the sample, A c is the control group absorbance, and A b is the blank absorbance.

HO·Scavenging Activity
An amount of 1.0 mL of a 1.865 mM 1,10-phenanthroline solution and 2.0 mL of the sample were added to a screw-capped tube and mixed. Then, 1.0 ml of a FeSO 4 ·7H 2 O solution (1.865 mM) was added to the mixture. The reaction was initiated by adding 1.0 mL of H 2 O 2 (0.03%, v/v). After incubating at 37 • C for 60 min in a water bath, the absorbance of the reaction mixture was measured at 536 nm against a reagent blank. The reaction mixture without any antioxidant was used as the negative control and a mixture without H 2 O 2 was used as the blank. The HO· scavenging activity was calculated using the following formula: HO· scavenging activity (%) = [(A s − A n )/(A b − A n )] × 100%, where A s , A n , and A b are the absorbance values determined at 536 nm of the sample, the negative control, and the blank after the reaction, respectively. 3.6.3. O − 2 · Scavenging Activity Superoxide anions were generated in 1.0 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1.0 mL of NADH (624 mM) and 1 mL of different sample concentrations. The reaction was initiated by adding 1.0 mL of phenazine methosulphate (PMS) solution (120 µM) to the reaction mixture. The absorbance was measured at 560 nm against the corresponding blank after a 5-min incubation at 25 • C. The O − 2 · scavenging activity was calculated using the following equation: where A c is the absorbance without sample and A s is the absorbance with sample.

Statistical Analysis
The data are expressed as the mean ± SD (n = 3). An ANOVA test using the SPSS 19.0 (Statistical Program for Social Sciences, SPSS Corporation, Chicago, IL, USA) was used to comparatively analyze