Evaluation of Antioxidant Activities of Extract from Beijing Roast Duck

Abstract

The aim of the study was to evaluate antioxidant activity of aqueous extract from Beijing roast duck through assessments of scavenging activity of 1,1-diphenyl-2-picrylhydrazil (DPPH) and 2,2′-azinobis(3-ethylbenothiazoline-6-sulphonic acid) diammonium salt (ABTS) and reducing power. Compared with non-roast duck, the antioxidant activity of roast duck extract was strongly improved. The different molecular weight compounds of ducks extracts were obtained through ultrafiltration, and fraction two (MW, 2 – 5 kDa) had the highest antioxidant capacity in all the four fractions. Furthermore, the roast duck extract was significantly found to increase the superoxide dismutase (SOD) activity and cell viability and to inhibit the malonaldehyde (MDA) content in the H₂O₂-treated Caco-2 cells, suggesting that the roast duck extract attenuated cellular oxidative damage. The fraction three and four (MW < 2 kDa) represented the strongly protective effect against cellular oxidative damage in the four fractions. Thus, the roast duck was considered to possess an effective antioxidant activity.

Introduction

Beijing roast duck is a special traditional recipe which has a history of more than 300 years originating from imperial kitchen, and it is famous for crispy skin, tender meat, glossy color ruddy, and attractive flavour. A standard procedure for Beijing roast duck can be separated into three major parts, including unhairing, clearing, and baking (Lin et al., 2011). Before duck is baked, maltose is usually daubed on skin of the duck. Baking is the key step to decide taste of duck; heat control and time must be mastered. Smokeless hardwood is used, temperature of the oven must be controlled at the scope from 230 to 300°C, and baking time must be controlled at 30 min (Chen et al., 2009).

Several studies have reported the safety assessment and aroma-active compounds of Beijing roast duck. Lin et al. (2011) have assessed polycyclic aromatic hydrocarbons as the major carcinogenic potential compounds for human being in some baked foods, including benzopyrene and cyclopenta pyrene. However, the intake of benzopyrene from Beijing roast duck could not exceed daily intake from vegetables. Chen et al. (2009) found that the identified aroma-active compounds of Beijing roast duck were likely products of different reactions, such as Maillard/Strecker associated reactions, oxidation of fatty acids, and degradation of amino acids. However, the bioactivities of Beijing roast duck have never been addressed in the previous studies.

Although vitamin C and other thermosensitive nutrients in duck are impaired during the roasting process, Maillard reaction should appear between proteins and sugar or oxidative lipids in high temperature. Similar to the condition of Beijing roast duck, Maillard reaction is also a non-enzymatic reaction in a high temperature. The Maillard reaction is an intricate reaction which produces various Maillard reaction products (MRPs), such as aroma compounds, ultraviolet-absorbing intermediates, and melanoidins (Kim and Lee, 2010). The generation of MRPs can modify the important food properties such as flavour, colour, and stability (Hwang et al., 2011). Notablely, antioxidant activities of MRPs have been well documented. The Maillard reactions generated from an amino acid-sugar model system produced some compounds with prominent antioxidant activity (Lertittikul et al., 2007). Kim and Lee (2009) evaluated antioxidant activity of MRPs derived from glucose/glycine, diglycine, and triglycine model systems. Jiang and Brodkorb (2012) determined antioxidant activity of Maillard reaction products from α-lactalbumin and β-lactoglobulin with ribose model system. Therefore, we presumed that the roast duck may exhibit the stronger antioxidant property, resulting from the formation of a large number of Maillard reaction products during baking.

Antioxidant is one kind of substances which can scavenge free radicals to slow down or prevent radical chain reaction. The free radicals are involved in regulation of some biochemical reactions, including protein kinase, gene transcription, protein synthesis, and biochemical flux. However, excessive free radicals can cause oxidative stress and attack normal cells to evoke many diseases (Stoia and Oancea, 2012). Hence, intake of food rich in antioxidant compounds has been expected to be effective in attenuating or preventing many diseases.

The antioxidant activities of extracts from vegetable food have widely been concerned (Ahn et al., 2008; Boonmee et al., 2011), but only a few reports evaluated the antioxidant capacities of animal food. Serpent et al. (2012) evaluated antioxidant capacity in the raw and cooked meat. However, the antioxidant activity of Beijing roast duck has never been documented. Therefore, the objective of this study was to assess antioxidant properties of the extract from Beijing roast duck through in vitro antioxidant evaluation including free radical scavenging and reducing power. Further, Caco-2 cells (a human colon cancer cell line) model was used to investigate protective effect against oxidative injury.

Materials and Methods

Ducks    Beijing roast duck and non-roast duck both purchased from restaurants of Quanjude in Shanghai, China. The bone was removed. The edible portion of ducks was cut into small pieces (ca. 0.5 cm³).

Chemicals    1,1-Diphenyl-2-picrylhydrazil (DPPH) was purchased from Wako Pure Chemical Industries, Ltd (Japan). 2,2′-Azinobis(3-ethyl-benothiazoline-6-sulphonic acid) diammonium salt (ABTS) was purchased from Shanghai Baoman Biological Technology Co., Ltd (Shanghai, China). Hydroxymethylfurfural (HMF, ≥ 99.0%) and furfural (≥ 99.0%) were of HPLC grade and obtained from Sigma-Aldrich Co (St. Louis, Mo, USA). All chemicals used were of analytical grade and were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), unless mentioned otherwise.

Preparation of extracts from ducks    The extracts were obtained by sequential extraction. The small pieces of ducks (1000 g) were submitted to ethyl ether (1500 mL) for 24 h under agitation at room temperature (25 ± 2°C), and then the mixture was filtered. After transferring the residue into vacuum drying machine for volatilizing ethyl ether, the residue was extracted with 1000 mL boiling distilled water for 2 hours. Subsequently, the filtered aqueous extracts were transferred to a freeze dryer (FD-80, Beijing Boyikang Instrument Co., Ltd., China) and dried to powder. The dried powder was weighted, and the yield of the extracts was determined by weight.

Ultrafiltration of the extracts from ducks    The extracts diluted solution was isolated by filtration (Gu et al., 2010). The solution was subjected to ultrafiltration, using an ultrafiltration cell model equipped with 5, 2, and 1 kDa nominal molecular mass cut-off membranes. Four fractions were collected and freeze-dried to powder. The powder was weighted. This process yielded four fractions: Fraction 1 (F1) (MW > 5 kDa), Fraction 2 (F2) (2 < MW < 5 kDa), Fraction 3 (F3) (1 < MW < 2 kDa), and Fraction 4 (F4) (MW < 1 kDa).

Determination of protein, carbohydrate, and browning    The contents of protein and carbohydrate in extracts were measured using colorimetric methods including dinitrosalicylic acid and Bradford G-250 (Dong and Yao, 2008). According to Kim and Lee (2010), browning of crude extract (roast duck and non-roast duck) was measured at 294 nm and 420 nm using a Shimadzu UV2600 spectrophotometer (Shimadzu, Tokyo, Japan), respectively. The concentration of crude extract was at 2.5 mg/mL.

HPLC determination of hydroxymethylfurfural and furfural    The HPLC system (Shimadzu, Kyoto, Japan) consisted a of LC-10AT liquid chromatography, a SPD-10A VP Plus UV-Vis Detector, a LC-10AT VP Plus Pump, a CTO-10AS VP Plus Column Oven, and a CBM-20A system controller.

HMF and furfural determination was accorded to method of Andrade et al. (2010) with some modifications. Briefly, each sample (crude extract from roast duck and non-roast duck, 100 mg) was suspended in a 10-mL centrifuge tube with 5 mL of distilled water. The tubes were shaken by vortex for 1 min and clarified with 0.25 mL of zinc acetate (30%, w/v) and potassium ferrocyanide (15%, w/v). Then the mixture was centrifuged at 5000 rpm for 10 min at 4°C, and the residue was further extracted using 2 mL of distilled water. All the supernatants were collected and the volume was made up to 10 mL with distilled water. HMF and furfural were analysed using HPLC with a linear gradient of isopropanol (5 – 65%, in 30 min) at a flow rate of 0.5 mL/min and C18 column (4.6 × 150 mm, 5 µm, Wondasil, Japan) at 32°C. The detection wavelength was at 280 nm, and injection volume was 5 µL.

Determination of DPPH radical scavenging activity    The DPPH radical scavenging activity was determined according to the method of Matsusaka and Kawabata (2010) with some modifications. A 5 mL solution of extracts was added into 5 mL of 6.0 × 10⁻⁵ M DPPH, and the reaction mixture stood in the dark at room temperature for 30 min. Absorbance of the mixture was measured at 517 nm with a spectrophotometer. The mixture of 5 mL distilled water and 5 mL solution of DPPH was used as the blank, and the mixture of 5 mL distilled water and 5 mL solution of extracts was used as the control. The percentage of DPPH radical scavenging was calculated as follows:

  

A_blank, A_sample, and A_control referred to the absorbance of blank, sample, and control respectively. All samples were analyzed in triplicate and vitamin C was used as the control.

Determination of ABTS radical scavenging activity    ABTS radical scavenging activity was measured by the method of Matsusaka and Kawabata (2010) with some modification. The ABTS radical was prepared by mixing 5 mL 7mM of ABTS solution and 88 µL 140mM of potassium persulphate solution, and then put the mixture at room temperature in the dark overnight. The mixture was diluted with ethyl alcohol to obtain an absorbance of 0.70 ± 0.02 at 734 nm. A 200 µL of aqueous extracts solution was added to 9.8 mL of diluted ABTS radical solution, and the mixture stood at room temperature for 0.5 h. The absorbance of the mixture was measured at 734 nm with the spectrophotometer. The mixture of 200 µL distilled water and 9.8 mL of diluted ABTS radical solution was used as the blank, and 200 µL aqueous extracts solution and 9.8 mL of distilled water was used as the control. The percentage of ABTS radical scavenging was calculated as follows:

  

A_blank, A_sample, and A_control referred to the absorbance of blank, sample, and control respectively. All samples were analyzed in triplicate and vitamin C was used as the control.

Determination of reducing power    The reducing power of extracts was determined according to the method of Gupta and Prakash (2008) with some modifications. The solution of extracts was added into mixture of 2.5 mL phosphate buffered saline (pH 6.6) and 2.5 mL of 1% potassium ferricyanide (K₃Fe (CN)₆). The reaction mixture was incubated in water bath at 50°C for 20 min, and then chilled and added 2.5 mL of 10% trichloroacetic acid. The mixture was then centrifuged at 800 rpm for 10 min. The supernatant obtained (5 mL) was mixed with 5 mL of distilled water and 1 mL of 0.1% FeCl₃. The absorbance was measured at 700 nm using the spectrophotometer. The reducing power was calculated by the increase in the absorbance at 700 nm as follows:

  

A_blank, A_sample, and A_control referred to the absorbance of blank, sample, and control respectively. All samples were analyzed in triplicate and vitamin C was used as the control.

Cell culture    Caco-2 cells were cultured in DMEM (Gibco/BRL) medium which was added 10% fetal bovine serum (Gibco/BRL), 100 U/mL of penicillin, and 100 µg/mL of streptomycin and were maintained at 37°C under a 5% CO₂ atmosphere. The medium was changed every 2 – 3 days (Kitts et al., 2012). After cultured 24 h, cells were plated in 96-multiwell culture plates at 1 × 10⁵ cells per well with 200 µL growth medium which contained crude extract and the four fractions (roast duck and non-roast duck, sample concentration was at 1 mg/mL), respectively. After fostering 24 h, the medium was added 0.5 mg/mL of methylthiazolyldiphenyl-tetrazolium (MTT) and incubated for 4 h at 37°C. Then dimethyl sulfoxide (DMSO) was added to dissolve the formazan formed. Cell viability was investigated using a microplate reader (Bio Rad, Model No.680) at 570 nm, and calculated as a percentage relative to the basal control cell samples.

Protective effect on cellular oxidative damage induced by hydrogen peroxide    To investigate protective potential of the extracts to the damaged cells, Caco-2 cells were treated with hydrogen peroxide (H₂O₂). Caco-2 cells plated in 96 or 12-multiwell culture plates with 200 or 1000 µL growth medium at 1 × 10⁵ or 5 × 10⁵ cells per well. After fostering 24 h, medium was discarded and the fresh with various concentrations of H₂O₂ was added, cellular viability was assessed by MTT using the microplate reader after cultured 2 h (Vidyashankar and Patki, 2010). And the moderate concentration was used to assess the protective ability of the extract on injured cells in the following experiments.

After the cells were cultured 24 h same as front, medium was discarded and the medium with H₂O₂ (30 µM) was added in each group. Two hours later, they were washed with fresh medium to omit H₂O₂. Subsequently, the cells were continued to culture in 200 or 1000 µL growth medium with the crude extract and the four fractions (each sample concentration was at 1 mg/mL), respectively. The control group was treated without H₂O₂ or extracts. MTT was used to determine the cell viability as described previously (Ju et al., 2012).

Measurement of malonaldehyde and superoxide dismutase    After treated as described previously, the cells in each group were washed with fresh medium three times, and the medium was removed. The cells were disrupted with the cells lysate obtained from Nanjing Jiancheng Bioengeering Institute (Nanjing, China), and the supernatants were stored at −80°C until assayed for the intercellular malonaldehyde (MDA) content and superoxide dismutase (SOD) activity. The MDA content was measured in light of thiobarbituric acid reactive substances, and the SOD activity was evaluated by water soluble tetrazolium salts method. The MDA content and the SOD activity in Caco-2 cells were assayed using corresponding assay kits which were obtained from Nanjing Jiancheng Bioengeering Institute (Nanjing, China), according to the directions.

Statistics analysis    All collected dates in triplicate were reported as mean ± standard deviation (SD). Significant differences were determined using Duncan's multiple-range test at p < 0.05. Data were analyzed by analysis of variance and using SPSS software (SPSS for Windows, 16.0, 2007, SPSS Inc., USA).

Results and Discussion

Composition and Maillard reaction markers of the extracts    Table 1 shows composition (protein and carbohydrate content) and yield of the extracts. The contents of protein and carbohydrate from non-roast duck were slightly higher than those from roast duck. This difference could be due to Maillard reaction during the roasting process, and the loss of protein and carbohydrate could generate MRPs. Browning at 294 nm and 420 nm is frequently applied to investigate the intermediate Maillard reaction compounds, and it is an important and obvious feature in Maillard reaction (Morales and Boekel, 1998). As shown in Table 2, the absorbance of the roast duck crude extract was higher than that of non-roast duck at 294 nm or 420 nm, and HMF and furfural were identified in roast duck extract. HMF and furfural are mainly formed through Amadori reaction in acid condition, HMF can be degraded to furfural in high temperature, so they are indicative of the development of Maillard reaction (Andrade et al., 2010). These results indicated that Maillard reaction could happen in the roasting process.

Table 1. Composition and yield of the extracts from ducks.

Samples	Composition (mg/g)		Yield (g/100 g)
	Protein	Carbohydrate
Roast duck
CE	240.4 ± 0.1b	17.6 ± 0.1b	9.5 ± 0.1e
F1	83.8 ± 0.5d	4.3 ± 0.1e	26.9 ± 0.2a
F2	72.6 ± 0.3f	4.2 ± 0.1e	24.6 ± 1.0b
F3	45.5 ± 0.8h	3.6 ± 0.1f	25.3 ± 0.1b
F4	38.1 ± 0.9i	3.2 ± 0.1g	20.1 ± 0.8c
Non-roast duck
CE	274.1 ± 1.3a	21.0 ± 0.2a	6.3 ± 0.1f
F1	87.1 ± 0.1c	6.2 ± 0.1c	17.6 ± 0.8d
F2	76.3 ± 0.4e	5.2 ± 0.1d	21.1 ± 0.9c
F3	55.3 ± 0.9g	4.4 ± 0.1e	25.3 ± 0.4b
F4	52.5 ± 0.6g	4.2 ± 0.1e	21.8 ± 0.8c

CE, crude extract

Different letters denote significant difference at p < 0.05.

Table 2. Maillard reaction markers of crude extracts from ducks.

Extract	HMF (mg/g)	Furfural (mg/g)	Browning
			294 nm	420 nm
Roast duck	0.025 ± 0.002	0.089 ± 0.004	2.32 ± 0.21a	0.31 ± 0.04a
Non-roast duck	ND	ND	1.36 ± 0.03b	0.18 ± 0.01b

ND, not detectable.

Different letters denote significant difference at p < 0.05.

Antioxidant activity of the extracts    In order to determine antioxidant activity of the extracts, DPPH and ABTS radicals and reducing power were chosen. When an antioxidant is added into the DPPH radical medium the purple colour will fade, because antioxidant molecules can scavenge DPPH free radicals and change them to a colourless product (Amarowicz et al., 2004). ABTS free radical is commonly used by food researchers, and the blue ABTS radical cation is converted back to its colorless neutral form during the reaction with antioxidants (Walker and Everette, 2009). The reducing power of a compound may use as a significant indictor of its potential antioxidant capability, and it was evaluated through the translation from Fe³⁺ to Fe²⁺ (Gupta and Prakash, 2008). Compared with no-roast duck, the IC₅₀ values of DPPH, ABTS, and reducing power of roast duck were significantly decreased by 19.8%, 26.9%, and 14.5%, respectively. The antioxidant abilities of roast duck were higher than that of the non-roast duck, but they were lower than the activity of vitamin C (Table 3). These results demonstrated that the antioxidant activity was improved after roasting.

Table 3. IC₅₀ values of antioxidant activities of extracts from ducks.

Samples	IC50 values (mg/mL)
	DPPH	ABTS	Reducing power
Roast duck
CE	2.06 ± 0.02d	2.25 ± 0.02d	2.60 ± 0.20b
F1	2.25 ± 0.02c	2.45 ± 0.04c	2.83 ± 0.06b
F2	0.99 ± 0.03f	1.08 ± 0.07g	1.25 ± 0.09f
F3	1.75 ± 0.09d	1.91 ± 0.03e	2.21 ± 0.02c
F4	1.30 ± 0.05e	1.42 ± 0.07f	1.64 ± 0.03e
Non-roast duck
CE	2.57 ± 0.07b	3.08 ± 0.09b	3.04 ± 0.03a
F1	2.48 ± 0.03b	3.16 ± 0.03b	3.12 ± 0.15a
F2	1.88 ± 0.11d	1.60 ± 0.12f	1.74 ± 0.09e
F3	3.15 ± 0.09a	3.99 ± 0.07a	2.72 ± 0.03b
F4	2.99 ± 0.01a	2.51 ± 0.06c	2.01 ± 0.04d
Vc	0.16 ± 0.03g	0.82 ± 0.06g	0.37 ± 0.03g

IC₅₀ value is the effective concentration at which DPPH and ABTS radicals were scavenged by 50% or for reducing power the absorbance was 0.5 at 700 nm.

CE, crude extract; Vc, vitamin C

Different letters indicate significant difference at p < 0.05.

Antioxidant activity of molecular-weight fractions through ultrafiltration    To investigate the further characteristic of the antioxidant activity of the ducks extracts, the molecular-weight fractions were obtained through ultrafiltration and were collected to determine the ABTS and DPPH scavenging activity and the reducing power. Four fractions were collected via ultrafiltration. According to the principle of ultrafiltration, the molecular weight distributions were: F1 > 5 kDa; F2, 2 – 5 kDa; F3, 1 – 2 kDa; F4 < 1 kDa. The marked differences were found among the fractions (roast duck and the non-roast duck) in the content of protein and carbohydrate (Table 1). The IC₅₀ values of four fractions (roast duck and non-roast duck) are shown in Table 3. The IC₅₀ values of DPPH, ABTS and reducing power of roast duck were found in the order of F1 > F3 > F4 > F2, but for non-roast duck, the order was F3 > F4 > F1 > F2 (DPPH), F3 > F1 > F4>F2 (ABTS), and F1 > F3 > F4 > F2 (Reducing power), respectively. F2 exhibited the highest in vitro antioxidant activity in both roast duck and non-roast duck, and antioxidant activity of the F2 of roast duck was significantly higher than that of non-roast duck. Generally, antioxidant activity was closely related to molecular weight. These results were similar to the previous report of Su et al. (2011), who found that a small and medium molecular weight (Mw < 5 kDa) of MRPs achieved a higher antioxidant activity.

There could be some antioxidant compositions in the duck extract, such as some proteins or peptides which have antioxidant property. Lee et al. (2012) reported that antioxidative peptide from fresh duck skin by-product protected liver against oxidative damage and it also shown high radical scavenging activity. Moreover, MRPs could be generated in the process of roasting duck, and a large number of studies have reported that MRPs had higher antioxidant activity. Zhao et al. (2013) had investigated antioxidant activity of HMF, and the results indicated that HMF could scavenge effectively ABTS and DPPH free radicals. Dragana et al. (2013) reported that β-lactoglobulin-glycoconjugates obtained by Maillard reaction in aqueous model systems under neutral condition showed higher radical scavenging activity. Dong et al. (2011) found that MRPs from casein peptide-glucose model increased free radical scavenging. In this study, we found a strong increase marker of MRPs in crude extract of the roast duck, and therefore, the increased antioxidant activities of roast duck extract should be closely related to MRPs.

Effect of the extract and fractions on cell viability    Before examining the protective effects of the crude extract and fractions on cell viability of the H₂O₂-treated Caco-2 cells, its potential cytotoxicity was tested. As shown in Fig. 1, compared with the control, the cell viability of the crude extract and four fractions was not decreased, suggesting that the dose of the extracts from ducks was not potential toxic to cells.

Fig. 1.

Viability of the Caco-2 cells cultured with crude extract (CE) and four fractions (F1, F2, F3, F4). The medium contained 1 mg/mL sample. Cell viability was determined by methyl thiazolyldiphenyl tetrazolium assay. Different superscript letters show significant difference at p < 0.05.

Protective effect on the cellular oxidative damage induced by H₂O₂    H₂O₂ is widely used as an oxidative stress in intracellular for its cellular action and its mechanism have been well studied (Sousa-Lopes et al., 2004) Caco-2 cells resemble closely the morphologically, and its functionally of the human small intestine epithelium and its sensitive to H₂O₂ (Manna et al., 1997). Therefore, Caco-2 cells model was used to evaluate the protective effect of ducks extracts against oxidative damage. Figure 2(a) demonstrates that the H₂O₂ induced growing injury in Caco-2 cells and that the higher concentration of H₂O₂, the higher the damage degree was. The concentration of H₂O₂ used in this study was range from 5 to 80 µM. When the concentration of H₂O₂ was 30 µM, the relative cellular viability was close to 50%. Hence, this concentration was moderate dose for evaluating the preventive effect. The injury of H₂O₂ to the cells was very serious, proteoglycans in the cell membrane was degraded by H₂O₂, and arachidonic acid was released. Then reactive oxygen entered into the cells and broke the balance between antioxidant defenses (antioxidant enzymes) and free radicals generation, which caused molecular damage and cell injury. In addition, H₂O₂ reacted with metal ions in cells to generate extremely highly toxic hydroxyl radicals through the Fenton reaction (Dan et al., 1996).

Fig. 2.

(a) H₂O₂ induced cytotocixity to the Caco-2 cells. The cells were cultured with H₂O₂ at various concentrations. (b) Protection ability of the crude extracts (CE) from ducks and fractions (F1, F2, F3, and F4) on the damaged cells induced by H₂O₂. The additional CE or fraction concentration in the medium was 1 mg/mL. The Caco-2 cells were incubated with 30 µM H₂O₂ except for the control group, and the control and H₂O₂ groups were cultured without duck extract. Cellular viability was determined using methyl thiazolyldiphenyl tetrazolium method. Different superscript letters denote significant difference at p < 0.05.

The results from chemical antioxidant indicated that the extract from roast duck had higher antioxidant activity than that form non-roast duck. Hence, to investigate further antioxidant bioactivity, we evaluated the protective effects of the extracts on H₂O₂-induced cell injury. Figure 2(b) shows that the cellular viability of the crude extract from non-roast duck, including the four fractions by ultrafiltration, was markedly lower than that from roast duck. The cellular viability in the crude extract from roast duck and its fractions (F1, F2, F3, and F4) groups was higher than that in the cellular oxidative damage group but lower than that in the blank, suggesting that the roast duck extract protected partially the cells growth against the injury of H₂O₂.

Cellular viability in the F3 and F4 (low molecular weight) groups (roast duck) was higher than that in the other fractions, which could be due to some produced bio-antioxidant compounds (low molecular weight) during the roasting process. However, these results were different from the in vitro evaluation through free radical scavenging. The F2 (MW, 2 – 5 kDa) had the highest antioxidant activity in vitro antioxidant assessment, but larger molecular compound could be different to be transported into cell through cytomembrane. Therefore, the smaller molecular fractions had stronger effective antioxidant capacity to engage in intracellular defense system against oxidative injure.

SOD activity and MDA content in the injured cell was determined to further evaluate the protective effect in the present study. The H₂O₂ treatment induced an increase in MDA content and a decrease in SOD activity in Caco-2 cells (Fig. 3). The roast duck extract significantly improved SOD activity and inhibit MDA generation compared with the non-roast duck extract, but did not fully resort to the control. The F3 and F4 (roast duck) had the more strongly preventive effect in the all fractions.

Fig. 3.

(a) Malonaldehyde (MDA) content and (b) Superoxide dismutase (SOD) activity in the Caco-2 cells. The additional crude extract (CE) or fraction (F1, F2, F3, and F4) concentration in the medium was 1 mg/mL. The Caco-2 cells were incubated with 30 µM H₂O₂ except for the control group, and the control and H₂O₂ groups were cultured without duck extract. Different superscript letters show significant difference at p < 0.05.

SOD is one kind of important intracellular antioxidant enzymes and catalyzes O₂⁻ to H₂O₂, and H₂O₂ is further catalyzed to O₂ and H₂O by catalase. Antioxidant enzymes are important to maintain cellular redox status which is required for normal cell growth and function (Muller et al., 2006). MDA is a direct index of lipid peroxidation and indirectly indicates the degree of cellular oxidative injured (Jin et al., 2013). The low SOD activity and high MDA level in the H₂O₂-induced injured cells may be because H₂O₂ broke the balance of redox status, influenced the signal pathway of protein expression, damaged DNA translation process, and subsequently resulted in oxidative damage (O'Sullivan et al., 2013). In this study, the roast duck extract increased cellular antioxidant capacity and consequently improved cellular defense against oxidative injury.

Some bioactive components, such as carotenoids, flavonoids, polyphenol, and fruit extracts, have been similarly reported to protect against oxidative damage in Caco-2 cells. The mechanism for the protective effect was attributed to the increasing antioxidant enzyme activities, increasing mitochondrial enzyme activities, or inhibiting apoptotic signaling pathway (Alemany et al., 2013; Cilla et al., 2008). We firstly reported that the roast duck extract attenuated oxidative damage in Caco-2 through increasing antioxidant enzyme (SOD) activities, and the further mechanism should be explored in the future studies. Additionally, MRPs has been recently known to protect the Caco-2 cells against the H₂O₂-induced injury and cell toxicity and to have effective recovery of antioxidant enzyme activity (Chuang et al., 2012). Ding et al. (2010) had evaluated protective effects of HMF on human hepatocyte LO2 injured by H₂O₂, and HMF may balance the functions of the caspase-3 related apoptosis to inhibit the cell apoptosis. Hence, MRPs in the roast duck extract could be important to engage in repairing oxidative damage.

In conclusion, this study examined antioxidant activities of the aqueous extract from Beijing roast duck through in vitro free radical scavenging and through oxidative stress model in Caco-2 cells. It is important to note that antioxidant activity of Beijing roast duck was stronger, which may be related to generation of some bioactive compounds (such as MRPs) during roast. Moreover, the roast duck extract attenuated the H₂O₂-induced cellular oxidative damage in Caco-2 cells. The isolated fractions had different antioxidant activities. F2 (MW, 2 – 5 kDa) of roast duck had the highest activity of in vitro free radical scavenging, and the F3 and F4 (low molecular weight) had the strongest protective effect against the H₂O₂-induced cell injury. In this sense, the present results demonstrated that Beijing roast duck could be beneficial to the human health. The more chemical structure information on bioactive components in the extract and its antioxidant mechanisms will be required in the further studies.

Acknowledgments    This study was supported by Sino-German Cooperation Project (GZ727).

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