Effect of Various Levels of Dietary Curcumin on Meat Quality and

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Effect of Various Levels of Dietary Curcumin on Meat Quality and Antioxidant Profile of Breast Muscle in Broilers Jingfei Zhang, Zhiping Hu, Changhui Lu, Kaiwen Bai, Lili Zhang, and Tian Wang* College of Animal Science and Technology, Nanjing Agricultural University, No. 6 Tongwei Road, Xuanwu District, Nanjing 210095, People’s Republic of China S Supporting Information *

ABSTRACT: The aim of this study was to determine the effects of curcumin on meat quality and antioxidant profile of breast muscle in broilers. In experiment 1, birds were fed basal diet with an additional 0, 50, 100, or 200 mg/kg curcumin, respectively. The results showed that dietary curcumin significantly increased the redness values of meat, catalase activity, and ABTS radical scavenging activity and decreased drip loss at 48 h. In experiment 2, birds reared under heat stress were assigned to similar treatments as experiment 1. Significant differences in the redox status of breast muscle were observed between the control and heat stress groups. The various levels of curcumin significantly prevented reactive oxygen species overproduction, enhanced the antioxidant defense system, and alleviated the abnormal change of antioxidant-related gene expression of muscle in heat-stressed birds. It was concluded that curcumin, as a potential antioxidant, improved meat quality and oxidant stability of muscle in broilers, whereas the inclusion of 50 and 100 mg/kg would be more efficient. KEYWORDS: curcumin, broiler, meat quality, free radicals, antioxidant status

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INTRODUCTION During the past few years, broiler chicks have become the fastest growing and most efficient meat species around the world.1 Compared to other edible meat, such as beef and pork, poultry meat is preferred by consumers due to its desirable nutritional characteristics.2,3 However, a high concentration of polyunsaturated fatty acids in poultry meat makes it very susceptible to oxidation, which negatively affects the meat quality and decreases the nutritional values.4 It has been proved, in living animals, that the antioxidant profile of tissues has a major impact on the oxidant stability of the resulting meat products.5 Studies revealed that enhanced antioxidant efficacy in broiler chickens will protect against free radical-induced lipid peroxidation chain reaction as well as protein oxidation in meat prior to and after slaughter. A common way to improve the antioxidant status of an organism is by dietary manipulation, such as antioxidant supplementation. Recently, natural antioxidants derived from herbs, spices, and plant extracts have received widespread attention considering the concerns about the safety of synthetic antioxidants and the increasing demand for natural and healthy food. Among these natural antioxidant sources, turmeric, the rhizomes of the plant Curcuma longa Linn., is widely consumed as a spice and traditional medicine in India, China, and Southeast Asia and has been authorized by the U.S. Food and Drug Administration (FDA) since 1994 as a coloring and flavoring agent in food.6 Curcumin, the major active component isolated from turmeric, exhibits the bright yellow color and has a variety of pharmacological properties, such as antioxidant, anti-inflammatory, and anticarcinogenic activities.7−9 Curcumin is a chain-breaking antioxidant and efficient in protecting animals against oxidant stress-induced damages.10 Work in our laboratory has led to the proposal that the antioxidant capacity of curcumin is mainly attributed to its © 2015 American Chemical Society

ability to directly scavenge free radicals and indirectly enhance the endogenous antioxidant defense system.11,12 Moreover, the protecting effect of dietary curcumin on the animal antioxidant system has been identified in red blood cells,12 liver,11,13 kidney,14 heart,15 intestine,16 muscle,17 and brain18 in vivo. One of these most exciting findings on curcumin research is the description of its capacity to protect the muscle from oxidation.19 High temperature is a major concern influencing the health and production of poultry.20 Heat stress promotes reactive oxygen species (ROS) overproduction, which is a well-known indicator of oxidant stress, influencing the yield of lean muscle tissue and impairing the meat quality.21 Mujahid et al. reported that the down-regulation of avian uncoupling protein (avUCP) under heat stress serves as a key contributor to the oxidation of meat in broilers.22 Studies showed that the negative effects of heat stress on meat quality in animals can be attenuated by dietary supplementation of antioxidants, such as vitamins, minerals, and natural extractions with antioxidant potential.23 However, little information is available regarding the effect of dietary curcumin on the meat quality and the antioxidant profile of breast muscle in broilers reared under heat stress. In the present study, the aims of this study were, first, to evaluate the effects of various levels of supplemental curcumin on meat quality and antioxidant profile of the breast muscle in broilers (experiment 1) and, second, to investigate whether dietary curcumin supplementation alleviated the oxidant damages and improved the antioxidant defense capacity of the breast muscle under chronic heat stress (experiment 2). Received: Revised: Accepted: Published: 3880

December March 22, March 30, March 30,

6, 2014 2015 2015 2015 DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886

Article

Journal of Agricultural and Food Chemistry Further research focused on the regulation of the antioxidantrelated gene by curcumin supplementation in experiment 2 was carried out to elucidate the possible mechanism by which supplemental curcumin protected the meat from oxidation under heat stress conditions.

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drip loss (%) =

Wbefore − Wafter × 100 Wbefore

where Wbefore is the initial weight of breast muscle sample and Wafter is the weight of breast muscle sample after 24 or 48 h of hanging. About 15 g of each breast muscle sample was weighed, held in plastic bags, and immersed in a water bath at 80 °C until the internal temperature reached 75 °C. Then, the bags were cooled to approximately 25 °C, carefully wiped, and reweighed.27 The cooking loss were calculated by applying the equation

MATERIALS AND METHODS

Ethics Statement. This experimental protocol was approved by the Ethical Committee and conducted under the supervision of the Institutional Animal Care and Use Committee of Nanjing Agricultural University, Nanjing, China. Reagents and Chemicals. 2,2-Dipheny-l-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and curcumin were purchased from Sigma Chemical Co., Ltd. All other chemicals used in the present study were obtained from Shanghai Chemical Agents Co., China, and were of analytical grade. Animals and Diets. Two experiments were done to investigate the beneficial effects of curcumin on the meat quality and redox system of breast muscle in broilers. In experiment 1, 320 1-day-old male broiler chicks (Arbor Acres, Gallus gallus domesticus) were obtained from a commercial hatchery (Hefei, Anhui, People’s Republic of China) and randomly allotted to four treatments, in which there were eight replicates and 10 birds per replicate. All of the birds were placed in wire cages in a three-level battery and kept in an environmentally controlled room maintained at 34−36 °C until the birds reached 14 days of age and then gradually decreased to 26 ± 1 °C by 21 days of age, after which it was maintained at room temperature (24−26 °C) until the end of the experiment. The birds in the control group (C1) were fed a basal diet (Supporting Information, Table S1), and the other three groups (C2, C3, and C4) were offered the experimental diets based on the basal diet with an additional 50, 100, or 200 mg/kg curcumin, respectively. Feed and water were provided ad libitum to all the birds throughout the 42 day experiment period. At the end of the experiment, one bird per replicate was randomly selected and killed for the left breast muscle samples. One part of the left breast muscle was stored at 4 °C for meat quality measurement (pH, color, drop loss, and cooking loss), and the remainder was stored at −80 °C for biochemical assays. In experiment 2, a total of 400 Arbor Acres broiler chickens were raised at a recommended environmental temperature from 1 to 20 days of age at the Qing Long Mountain Animal Center, Nanjing, China. At 21 days of age, broilers with similar body weight (BW) were randomly allocated into five treatment groups with eight replicate pens of 10 birds per pen, T1, T2, T3, T4, and T5. Birds in the T1 and T2 groups received the basal diet (Table S1), whereas birds in the T3, T4, and T5 groups received the basal diet containing 50, 100, or 200 mg/ kg curcumin, respectively. Birds from the T1 group were housed at 24 °C for 24 h/day, whereas birds from the T2, T3, T4, and T5 groups were housed at 34 °C for 8 h/day (9:00 a.m.−5:00 p.m.) followed by 22 °C for 16 h/day. All of the broiler chickens had free access to water and feed and enjoyed a 12 h light−dark cycle of light regimen during the whole experiment. After 21 days of heat exposure, birds from each replicate were slaughtered and breast muscle examples were collected for homogenate preparation and real-time PCR analysis. Meat Quality Measurement. The breast muscle pH was measured at 45 min (pH45) and 24 h (pH24) post-mortem using a pH meter (PH-STAR, Mattuas, Germany), as described by Zhen.24 Three measurements were taken from each sample. The color of the breast muscle was measured with a colorimeter (Minolta CR-10, Konica Minolta Sensing, Japan), based on the CIE L*a*b* system in which the L*, a*, and b* values were determined as indicators of lightness, redness, and yellowness, respectively.25 The average of the three measurements was recorded as color coordinate values of the sample. For estimation of drip loss, breast muscle samples, size of 3 cm (length) × 2 cm (width) × 1 cm (thickness), were stored at 4 °C for 24 h post-mortem. The breast sample was placed in netting and suspended in a vacuumed bag for 24 and 48 h at 4 °C, respectively.26 The drip loss of the breast muscle was calculated using the equation

cooking loss (%) =

Wbefore − Wafter × 100 Wbefore

where Wbefore is the initial weight of breast muscle sample and Wafter is the weight of breast muscle sample after heating. Preparation of Breast Muscle Homogenates. The minced breast muscle was homogenized in ice-cold 0.86% sodium chloride buffer (w/v, 1:9) and centrifuged at 3500g for 10 min at 4 °C. The supernatant was stored at −80 °C for further analysis. The protein concentrations of the 10% breast muscle homogenates were determined by using the bicinchoninic acid assay.28 Measurement of Antioxidant Potential. DPPH, ABTS+, and superoxide radical (O2−) radical scavenging activities were estimated according to the methods described in our previous study.12 Hydroxyl radical (OH−) radical scavenging assay was determined according to the method by Noda.29 The percentage inhibition of OH− generation was calculated using the formula

OH− radical scavenging effect (%) =

Acontrol − A sample Acontrol

× 100

where Acontrol is the absorbance of the control reaction and Asample is the absorbance of the test samples. Determination of Protein Carbonyls (PC) and 8-Hydroxy-2′deoxyguanosine (8-OHdG). The PC levels in the breast muscle were determined using 2,4-dinitrophenylhydrazine as described by Reiter.30 The PC content was expressed as nanomoles per milligram of protein. The level of 8-OHdG in the breast muscle was determined by using an ELISA kit (Beijing North Institute of Biotechnology, Beijing, China) with a monoclonal antibody specific for broiler species. The procedure followed the manufacturer’s protocols. The 8-OHdG content was calculated by a standard curve generated using 0.05−2 ng/mL 8-OHdG and corrected for the protein concentrations in each breast muscle homogenate sample. The final results were expressed in nanograms per milligram of protein. Determination of Reactive Oxygen Species. Intracellular ROS production in breast muscle was measured by using 2,7-dichlorofluorescein diacetate as fluorescence probe, as previously described.11 The fluorescence was measured at the excitation wavelength of 488 nm and an emission wavelength of 525 nm through a fluorescence spectrometer. The result was expressed as a percentage of the control group, which was taken as 100%. Determination of Total Thiol Contents. The concentration of total thiol groups (T-SH) was determined according to the method of Tabassum.31 The T-SH content was expressed as micromolers per milligram of protein. Determination of Antioxidant Enzyme Activity. The activities of total SOD (T-SOD), glutathione peroxidase (GPx), glutathione Stransferase (GST), and catalase (CAT) and the contents of malondialdehyde (MDA) and reduced glutathione (GSH) were determined spectrophotometrically with the commercial kits purchased from Nanjing Jiancheng Insititute of Bioengineering (Nanjing, Jiangsu, People’s Republic of China).32 The results of T-SOD, MnSOD, GPx, GST, and CAT activities were expressed as units (U) per milligram of protein. The GSH content was expressed as micrograms per gram of protein, and MDA content was expressed as nanomoles per milligram of protein. Real-Time Polymerase Chain Reaction (PCR) Analysis. Total RNA from the breast muscle was isolated using Trizol reagent (TaKaRa, Dalian, China). The RNA was quantified with a 3881

DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886

Article

Journal of Agricultural and Food Chemistry Table 1. Effect of Curcumin on Meat Quality and Antioxidant Status of Breast Muscle in Broilers (Experiment 1)a p value

curcumin treatment meat quality pH45 pH24 L* a* b* drip loss (%) at 24 h drip loss (%) at 48 h cooking loss (%) antioxidant status CAT (U/mg protein) T-SOD (U/mg protein) GPx (U/mg protein) GST (U/mg protein)

C1b

C2

C3

C4

SEM

linearc

quadratic

6.05 5.89 44.33 4.45 c 13.16 5.33 6.91 a 13.95

6.04 5.88 44.74 4.93 bc 13.35 4.63 6.30 ab 13.49

6.11 5.86 44.56 5.69 ab 13.88 4.13 5.36 b 13.40

6.15 5.88 45.05 5.89 a 13.05 4.92 6.22 ab 13.68

0.023 0.016 0.181 0.177 0.310 0.182 0.180 0.367

0.057 0.873 0.224 0.001 0.949 0.287 0.059 0.789

0.146 0.895 0.480 0.003 0.740 0.062 0.016 0.859

2.46 b 60.73 30.34 31.78

3.04 ab 84.03 34.48 37.45

4.36 a 64.60 39.71 34.09

2.03 b 65.49 32.60 32.78

0.297 2.648 1.538 1.481

0.987 0.832 0.392 0.977

0.043 0.101 0.128 0.515

a

Values in the same row followed by different letters are significantly different (n = 8). bC1 group, basal diet; C2 group, basal diet containing 50 mg/ kg curcumin; C3 group, basal diet containing 100 mg/kg curcumin; C4 group, basal diet containing 200 mg/kg curcumin. cOrthogonal polynomials were used to investigate linear and quadratic responses to the level of curcumin treatment.

Table 2. Effect of Curcumin on Free Radical Scavenging Activity of Breast Muscle in Broilers (Experiment 1)a p value

curcumin treatment b

DPPHd (%) ABTS (%) O2− (%) OH− (%)

c

C1

C2

C3

C4

SEM

linear

quadratic

22.22 31.37 c 16.39 34.02

25.96 36.45 b 23.23 35.03

31.40 42.04 a 24.84 36.10

26.22 32.93 c 19.14 37.20

0.603 0.246 1.263 0.556

0.907 0.035 0.401 0.205

0.745 0.004 0.186 0.353

a

Values in the same row followed by different letters are significantly different (n = 8). bC1 group, basal diet; C2 group, basal diet containing 50 mg/ kg curcumin; C3 group, basal diet containing 100 mg/kg curcumin; C4 group, basal diet containing 200 mg/kg curcumin. cOrthogonal polynomials were used to investigate linear and quadratic responses to the level of curcumin treatment. dThe free radical scavenging activity was calculated on the basis of the protein content (mg/mL) in breast muscle. spectrophotometer (NanoDrop 2000c, Thermo Scientific, USA) and tested by agarose gel electrophoresis. Reverse transcription was performed immediately following the RNA isolation using the Perfect Real Time SYBR PrimeScriP kit (TaKaRa). Then the cDNA samples were amplified with the SYBR Premix Ex TaqII Tli RNaseH Plus kit (TaKaRa). Primer sequences are listed in Table S2. The relative amount of each target gene mRNA was calculated using the 2−ΔΔCt method. The expression level of each target gene mRNA was normalized to the mRNA level of β-actin. In the present study, the results of gene expression were expressed as the fold changes between the treated and control groups. Statistical Analysis. All data (experiments 1 and 2) were analyzed by using one-way analysis of variation (ANOVA) by SPSS 17.0. To evaluate the difference among groups, multiple comparisons were conducted using the Duncan test. Linear and quadratic contrasts were used to determine the effects for different dose levels (0, 50, 100, or 200 mg/kg) of supplemental curcumin in experiment 1. The significance level for difference was p < 0.05.

The drip loss at 48 h of breast muscle was significantly influenced (p < 0.05) by dietary supplementation with curcumin and displayed a quadratic dose response. However, there was no significant effect on drip loss at 24 h and cooking loss of the breast muscle following the curcumin treatment. Table 1 also shows the effects of dietary curcumin supplementation on the levels of antioxidant enzymes in breast muscle of broilers. The CAT activities of the breast muscle in curcumin-supplemented groups showed a quadratic dose response (p < 0.05). However, the breast muscle GPx, TSOD, and GST activities of broilers fed the curcumin diet were not different (p > 0.05) from those of the broilers fed the basal diet. Minimal research has been conducted regarding the effect of various levels of curcumin on meat quality and oxidative status of breast muscle in broilers. Poultry meat contains a high concentration of polyunsaturated fatty acids, which makes it sensitive to free radical attack and oxidative deterioration.33 The oxidant reaction initiated by free radicals destroyed the normal structure of the muscle and accumulated deleterious byproducts, eventually influencing the meat quality.34 On the other hand, the oxidation of myoglobin, which was mainly responsible for the meat color post-mortem, could lead to the discoloration of meat.35 In the present study, curcumin, similar to fat-soluble antioxidant vitamin E, significantly improved the antioxidant capacity of the breast muscle. Similar beneficial effects of curcumin on enzyme activities were found in the literature.10,34 It was suggested that curcumin improved the meat quality, especially the meat color, primarily due to its

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RESULTS AND DISCUSSION Meat Quality and Antioxidant Status (Experiment 1). The effects of dietary curcumin on meat quality in breast muscle are presented in Table 1. Diet administered with curcumin had no difference on either the pH45 or pH24 of breast muscle in broilers. For meat color, the a* value in breast muscle showed a linear and quadratic increase (p < 0.05) following the dietary curcumin supplementation (p < 0.05), but the L* and b* values showed no significant difference among the treatment groups. The water-holding capacity in the breast muscle of broilers was expressed by the drip loss at 24 and 48 h. 3882

DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886

Article

Journal of Agricultural and Food Chemistry

Figure 1. Effect of curcumin and heat treatment on (A) intracellular ROS levels and the concentrations of (B) MDA, (C) PC, and (D) 8-OHdG in breast muscle (experiment 2). Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). T1 group, basal diet + normal temperature; T2 group, basal diet + heat treatment; T3, 50 mg/kg curcumin + heat treatment; T4, 100 mg/kg curcumin + heat treatment; T5, 200 mg/kg curcumin + heat treatment.

production.23,41 A high level of free radicals caused oxidant stress and triggered oxidation of some critical cellular biomolecules, including lipid, protein, and DNA. In the present study, Figure 1 shows the increased (p < 0.05) intracellular ROS (Figure 1A), MDA (Figure 1B), PC (Figure 1C), and 8OHdG (Figure 1D) levels in chickens reared under chronic heat treatment, suggesting the severe oxidant damage in breast muscle. When compared to the heat-stressed group, intracellular ROS levels in curcumin-administered groups (50, 100, or 200 mg/kg) were significantly decreased (p < 0.05) with the minimum level at 50 mg/kg. Compared with the heat-stressed group, administration with 100 mg/kg curcumin significantly decreased (p < 0.05) the breast muscle MDA and PC levels by 16.54 and 8.24%, respectively. In addition, curcumin supplementation (50, 100, or 200 mg/kg) significantly decreased the level of breast muscle 8-OHdG, which was a quantitative indicator for DNA damage occurring during oxidant stress.42 Curcumin had a strong capability of quenching free radicals, such as superoxide radicals and hydrogen peroxide.12 Studies reported that Cr(VI) induced an increase in ROS level and severe oxidant damage, which were remarkably blocked by curcumin administration.14 Besides, as a chain-breaking antioxidant, curcumin was proven to stop the reaction of chain propagation of lipid peroxidation through intercepting radical oxidation initiators.14 A similar result was obtained from the recent work of Waseem et al., which suggested that the protective effects of curcumin against oxidant damage were partly attributed to its excellent free radical scavenging activity.43 Effect of Curcumin on Antioxidant Defense System Damaged by Heat Stress (Experiment 2). To evaluate whether dietary curcumin administration attenuated the oxidant damage induced by chronic heat treatment, we determined the capacity of the antioxidant defense system, consisting of the nonenzymatic and enzymatic antioxidant. Results showed that there existed significantly decreasing (p < 0.05) T-SH and GSH concentrations in breast muscle after

ability to increase the antioxidant enzymatic activities in breast muscle. Free Radical Scavenging Activity (Experiment 1). The DPPH radical is a stable nitrogen radical with yellow color and can be dissolved only in organic media.36 The ABTS radical is a metastable radical with blue color and can be solubilized in both aqueous and organic media.37 Both of them have been widely used to measure the antioxidant activity of biological materials and characterized by excellent reproducibility and stability under certain assay conditions.38 O2− is one of the most representative free radicals, and its negative effects can be magnified because it produces other kinds of cell-damaging free radicals and oxidizing agents.39 OH− is well-known to be the most reactive of all the reduced forms of dioxygen and harmful to several cellular components such as DNA, lipid, and nucleus.29 The antioxidant capacities of the breast muscle in broilers measured as DPPH, ABTS, O2−, and OH− radical scavenging capacities are shown in Table 2. A linear and quadratic increase (p < 0.05) in the ABTS radical scavenging activities of breast meat was found with increasing dietary curcumin supplementation. The ABTS radical scavenging activities of breast meat in 50 and 100 mg/kg curcumin groups were significantly higher (p < 0.05) than that of the control group, whereas no difference was observed between the control group and the 200 mg/kg curcumin group. However, there was no significant variation (p < 0.05) in the percentage inhibition of DPPH, O2−, and OH− radicals in the breast meat from the curcumin supplemental groups as compared to the control group. The high efficiency of antioxidants in the free radical scavenging assays could be attributed to either its direct capacity to neutralize stable free radicals or an indirect role as a hydrogen donor.40 Our results suggested that curcumin, as a potential antioxidant, improved the antioxidant properties of breast muscle in broiler, possibly by the enhanced hydrogendonating ability and free radical scavenging capacity. Biomarkers of Oxidant Stress-Induced by Heat Treatment (Experiment 2). Studies on heat stress showed that its adverse effects are closely associated with the excessive ROS 3883

DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886

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Journal of Agricultural and Food Chemistry Table 3. Changes in Breast Muscle Antioxidant Enzyme Activity (Experiment 2)a T-SH (μmol/mg protein) GSH (μg/g protein) GPx (U/mg protein) GST (U/mg protein) CAT (U/mg protein) T-SOD (U/mg protein)

T1b

T2

T3

T4

T5

41.16 ± 0.54 a 24.18 ± 0.33 a 30.40 ± 0.50 a 31.68 ± 1.34 a 2.49 ± 0.03 a 64.14 ± 0.83 a

33.26 ± 1.09 d 17.84 ± 0.25 c 23.79 ± 0.25 c 23.04 ± 0.79 d 1.82 ± 0.02 d 47.77 ± 0.91 c

37.70 ± 0.67 b 20.66 ± 0.86 b 26.31 ± 0.34 b 28.83 ± 0.60 b 2.06 ± 0.07 bc 57.41 ± 3.53 b

36.47 ± 0.50 bc 21.36 ± 0.79 b 26.36 ± 0.30 b 26.88 ± 0.68 bc 2.15 ± 0.08 b 58.81 ± 1.77 ab

35.10 ± 0.60 cd 19.92 ± 0.91 b 25.30 ± 0.71 b 24.66 ± 0.73 cd 1.97 ± 0.06 cd 50.14 ± 0.57 c

a

Values are expressed as group mean values and SEM; mean values followed by different letters are significantly different. Test Duncan p < 0.05. bT1 group, basal diet + normal temperature; T2 group, basal diet + heat treatment; T3, 50 mg/kg curcumin + heat treatment; T4, 100 mg/kg curcumin + heat treatment; T5, 200 mg/kg curcumin + heat treatment.

exposure to heat treatment (Table 3). The T-SH level of chicken supplemented with 50 or 100 mg/kg curcumin was significantly increased by 13.35 and 9.65%, respectively, as compared with the heat-stressed group. The reduction of breast muscle GSH concentrations was attenuated to a significant extent upon curcumin supplementation (50, 100, or 200 mg/ kg), but not dose-dependently (p < 0.05). Moreover, the weakened antioxidant defense system in breast muscle was also evidenced by the remarkable reductions (p < 0.05) of GPx, GST, CAT, and T-SOD activities following the exposure to heat treatment (Table 3). Dietary treatment with curcumin significantly attenuated (p < 0.05) heat stress-induced decrease of GPx activity; notably the greatest activities were found at 100 mg/kg curcumin. Chickens supplied with 50 or 100 mg/kg curcumin had 25.13 and 16.67% higher (p < 0.05) GST activities than those of the heat-stressed group, respectively. A similar observation (p < 0.05) was made regarding the breast muscle CAT and T-SOD activities. The change of intracellular redox status is relevant to the ability of curcumin to cope with oxidative stress during heat exposure. To restore the balance of redox system caused by oxidant stress, cells facilitated the antioxidant defense system through enhancing activities of enzymatic antioxidants (CAT, T-SOD, and GSH-related enzymes) and elevating concentrations of nonenzymatic antioxidants (total thiols and GSH).44 The present study showed that GPx, GST, CAT, and T-SOD activities were significantly increased in the breast muscle with the curcumin administration compared with the heat-stressed group. In cells, SOD served as the first line of defense against deleterious effects of ROS via catalyzing the dismutation of endogenous superoxide radicals to H2O2, which is further removed by CAT and GPx.45 GST, a major group of GSHrelated enzymes, was responsible for the detoxification of endogenous compounds to reduce the cytotoxicity of ROS.46 Furthermore, T-SH and GSH levels were significantly increased in curcumin-supplemented groups. Generally, T-SH consists of nonprotein and protein-bound thiols, whereas GSH represents about 90% of the intracellular nonprotein thiols. Consistent with our results, numerous studies have confirmed that the depletion of GSH resulting from oxidant challenge was partially or even fully recovered with curcumin treatment.11,18 These above results indicated that curcumin had prominent antioxidant activity not only through its enhancement of antioxidant enzyme activities but also through its effect on the redox status of intracellular thiols, especially GSH. Gene Expression (Experiment 2). In the present study, birds reared in chronic heat stress showed typical characteristics of heat stress, including the overexpression of heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) mRNA in the breast muscle. As shown in Figure 2, curcumin

Figure 2. Effect of curcumin and heat treatment on gene expression in breast muscle (experiment 2). Results are expressed as the mean ± SEM. Bars labeled with different letters are significantly different (p < 0.05). T1 group, basal diet + normal temperature; T2 group, basal diet + heat treatment; T3, 50 mg/kg curcumin + heat treatment; T4, 100 mg/kg curcumin + heat treatment; T5, 200 mg/kg curcumin + heat treatment.

supplementation (50, 100, or 200 mg/kg) resulted in significant decreases in HSP70 and HSP90 expression levels. HSP70 and HSP90 are stress-defense proteins characterized by overexpression in response to many adverse environmental or physiological stimuli, including heat stress. Similar to our results, Hao et al. found a quick increase of HSP70 mRNA expression when chickens were exposed to heat stress for 2 h.47 Parallel to the results of Sahin et al., our results have ascertained the protective role of curcumin by suppressing HSP expression in heat-stressed birds.10 Furthermore, the mRNA expression of avUCP, an important regulator of mitochondrial ROS production,48 was obviously depressed on exposure to chronic heat treatment while being markedly stimulated by curcumin supplementation (50, 100, or 200 mg/kg). The above data were consistent with the result of ROS level in breast muscle, indicating the inhibitory effect of curcumin on ROS production under heat stress. Moreover, we found that chronic heat treatment also significantly down-regulated sirtuin 1 (SIRT1) mRNA levels. SIRT1, an NAD+-dependent protein deacetylase, plays an important role in the regulation of cellular oxidative stress burden.49 Studies reported that the SIRT1 activation significantly decreases ROS levels, either directly or indirectly by deacetylation of the forkhead box class O 3 transcription factor and induction of manganese superoxide dismutase.50 Recently, Yang et al. identified that SIRT1 activation by curcumin efficiently attenuated oxidant damage in the isolated heart.15 In the present study, we found that the muscle mRNA expression of SIRT1 in heat-stressed groups, although not 3884

DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886

Article

Journal of Agricultural and Food Chemistry

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significant, tended to increase in response to dietary curcumin treatment (100 or 200 mg/kg). In conclusion, this study demonstrated that curcumin could be a potential feed additive that increased the oxidant stability of muscle and the meat quality in broiler chickens. Dietary curcumin supplementation could improve the meat color and increase water-holding capacity, free radical scavenging activity, and antioxidant enzymatic activity. The inclusion of 50 or 100 mg/kg curcumin was more effective in improving the meat quality and antioxidant system of breast muscle in broilers. The various levels of dietary curcumin had a positive effect on the redox status of breast muscle in broilers reared under chronic heat stress. Therefore, these results demonstrated that curcumin could serve as a potential promising antioxidant in poultry production that improved the redox status of breast muscle, in both normal and heat-stressed conditions, and benefit the resulting meat quality.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed table of ingredient composition and calculated nutrient content of the basal diets and detailed table of primers used for real-time PCR (experiment 2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(T.W.) E-mail: [email protected]. Phone: +86 025 84395156. Fax: +86 025 84395156. Funding

This study was funded by the National Basic Research Program of China (2012CB124703) and Kehu Biotechnology Research Center, Guangzhou, People’s Republic of China. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful for the valuable comments made by anonymous reviewers and thank Yuxiang Yang and Chunlong Mu for assistance in the experiment and editing of the manuscript.

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ABBREVIATIONS USED ABTS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; avUCP, avian uncoupling protein; CAT, catalase; DPPH, 2,2-dipheny-l-picrylhydrazyl; GPx, glutathione peroxidase; GSH, reduced glutathione; GST, glutathione Stransferase; HSP70, heat shock protein 70; HSP90, heat shock protein 90; MDA, malondialdehyde; PC, protein carbonyls; O2−, superoxide radical; OH−, hydroxyl radical; ROS, reactive oxygen species; SIRT1, sirtuin 1; T-SH, total thiol groups; TSOD, total superoxide dismutase; 8-OHdG, 8-hydroxy-2′deoxyguanosine

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DOI: 10.1021/jf505889b J. Agric. Food Chem. 2015, 63, 3880−3886