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Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens
Animal Feed Science and Technology 163 (2011) 59–66
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Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens S.Ting, H.S. Yeh, T.F. Lien ∗ Department of Animal Science, National Chiayi University, 300 University Road, Luh Liau Li, Chiayi 600, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 10 July 2009 Received in revised form 30 September 2010 Accepted 4 October 2010
Keywords: Hesperetin Naringenin Egg quality Serum traits Antioxidant activity Laying hens
a b s t r a c t Hesperetin and naringenin phytochemicals are naturally occurring flavanoids in citrus fruits. The purpose of this study was to evaluate the effects of supplementing different levels of extracted hesperetin and naringenin on egg quality, serum traits and antioxidant activity in laying hens. Two experiments were conducted, each for 10 weeks, in a completely randomized experiment design. Each had 100 Leghorn laying hens (26 weeks old) randomly assigned into five groups (n = 20) based on dietary categories of hesperetin 0, 0.5, 1, 2, 4 g/kg and naringenin 0, 0.5, 1, 2, 4 g/kg. Experimental results indicated that there was increased (P<0.05) egg production in the 1 g/kg naringenin-supplemented group, but lower (P<0.05) egg production in the hesperetin- and naringenin-supplemented groups given 4 g/kg. Cholesterol content (per gram yolk) and total cholesterol content (per egg) were lower (P<0.05) in the hesperetin- and naringenin-supplemented groups as compared to the control group, and the 2 g/kg hesperetin- and naringenin-supplemented groups showed the most significant difference. Both serum cholesterol and triglyceride concentrations were lower (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups. The SOD and catalase activities, scavenging O2 − and iron-chelating abilities were higher (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups, and the trolox equivalent antioxidant capacity was higher (P<0.05) in the 2 g/kg naringenin-supplemented group. The results confirmed that both hesperetin and naringenin could lower serum and egg yolk cholesterol levels, and improve the antioxidant activities, however the measured variables generally showed significant quadratic responses to increasing amounts of the compounds. The recommended supplementation level of hesperetin and naringenin is 2 g/kg of the basal diet for reduced serum and yolk cholesterols contents and increased antioxidant capacity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Eggs have a high biological value and contain a wide array of nutrients needed by the human body to maintain good health. Eggs have been shown to be a major source of dietary cholesterol (180–250 mg) (Elkin, 2009). However, dietary cholesterol significantly elevates plasma cholesterol in about 15–30% of the population (hyper-responders) (Elkin, 2006). Thus, some organizations, such as the American Heart Association and the National Cholesterol Education Program, recommend a population wide limitation of dietary cholesterol intake to less than 300 mg/day (Krauss et al., 2000; National Cholesterol Education Program, 2002). One of the dietary recommendations in the prevention of coronary heart disease for example is to limit egg consumption. Thus, demand for a lower cholesterol concentration has led to many attempts to reduce
∗ Corresponding author. Tel.: +886 5 2717536; fax: +886 5 2750134. E-mail address: tfl[email protected] (T.F. Lien). 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.10.001
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Table 1 Composition of basal diets (as feed basis). Ingredients
g/kg
Yellow corn meal Soybean meal, 445 g crude protein/kg Fish meal, 600 g crude protein/kg Soybean oil Dicalcium phosphate Limestone, pulverized Sodium chloride Vitamin premixa Mineral premixb Oyster shells dl-Methionine Choline chloride, 50 g/kg
607.5 240.0 30.0 20.0 13.0 42.5 3.0 0.5 2.0 40.0 0.5 1.0
Total
1000.0
Calculated value: Crude protein (g/kg) ME (MJ/kg) Calcium (g/kg) Available phosphorous (g/kg) Lysine (g/kg) Methionine + cystine (g/kg)
179 11.8 34.0 4.7 9.8 6.2
Analyzed value: Hesperitin (g/kg) Naringenin (g/kg)
Trace Trace
a Vitamin premix supplied the following per kilogram of diet: Vitamin A, 12,500 IU; Vitamin D3 , 3125 ICU; Vitamin E, 37.5 IU; Vitamin K3 , 6.25 mg; Vitamin B1 , 3.75 mg; Vitamin B2 , 12.5 mg; Vitamin B6 , 10.0 mg; Pantothenate, 18.8 mg; Niacin, 50 mg; Biotin, 0.06 mg; Folic acid, 1.25 mg; Vitamin B12 , 0.05 mg. b Mineral premix supplied the following per kilogram of diet: Cu (CuSO4 ·5H2 O, 25.45% Cu) 6 mg; Fe (FeSO4 ·7H2 O, 20.29% Fe) 50 mg; Mn (MnSO4 ·H2 O, 32.49% Mn) 40 mg; Zn (ZnO, 80.35% Zn) 60 mg; Se (NaSeO3 , 45.56% Se) 0.075 mg.
the cholesterol concentration in eggs. This has involved a number of different approaches, including genetic selection, avian transgenesis, nutritional interventions such as n-3 fatty acids (Elkin, 2006), non-nutritive dietary factors such as oxysterol (Elkin, 2007), and (or) pharmacological manipulation (Elkin, 2007). Hesperetin and naringenin and are naturally occurring flavonoids that can be extracted from the peels of oranges (Citrus sinensis) and grapefruits (Citrus × Paradisi). Flavonoids have exhibited a variety of biological and pharmacological activities, including the inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and Acyl-CoA: cholesterol acyltransferase (ACAT) enzymes (Bok et al., 2000; Kim et al., 2004), free radical scavenging, anti-inflammation, and inhibition of tumor promotion (Friedman, 2007; Yang et al., 2008; Seo et al., 2003). The principal citrus flavonoids, hesperetin from oranges and naringenin from grapefruits are structurally similar to genistein which has been tested as a potential agent for improving the cholesterol metabolism in diet-induced hypercholesterolemic animals (Kurowska et al., 1999). Hesperetin and naringenin have also been found to exhibit estrogenic, anti-carcinogenic (Erlund et al., 2001), and strong antioxidant functions (Jeon et al., 2001). A previous study by Lien et al. (2008) indicated that adding hesperetin and naringenin at 0.5 g/kg lowered serum and yolk cholesterol, and improved antioxidant ability. However, the work of Lien et al. (2008) only used an inclusion level of 0.5 g/kg and it is possible that the optimum inclusion level is above this amount. Therefore, the aim of the present study was to investigate the effects of supplementing graded levels of hesperetin or naringenin on egg quality, serum traits and antioxidant activity of laying hens, and assess whether these responses behave in a linear or quadratic manner. 2. Materials and methods 2.1. Experimental design and measurements Two experiments were conducted simultaneously; each used 100 individually-caged, single-comb white Leghorn laying hens (26 weeks old) randomly assigned into 5 groups (n = 20) and fed with diets containing hesperetin at 0, 0.5, 1, 2, 4 g/kg or naringenin at 0, 0.5, 1, 2, 4 g/kg. Hens were each fed with about 110 g diet every day, water was supplied ad libitum and lighting was controlled (15 h) throughout the 10-week experimental period. Eggs were collected daily and egg production was recorded. Four eggs per hen per treatment were collected during the end of the experiment. Blood (about 10 mL) was collected (from brachial wing vein) in a glass tube (16 mm × 100 mm) at the end of the experiment, blood samples were left to stand at room temperature for clotting, and then the serum was obtained by centrifugation at 1500 × g for 10 min. The composition of the basal diets for both experiments is shown in Table 1. This study was approved by the Animal Care and
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Use Committee of the University. Animals used in this experiment were cared for under the guidelines stated in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. 2.2. Hesperetin and naringenin extraction method The hesperetin and naringenin extraction from peels followed the method described by El-Nawawi (1995). Orange peel or grapefruit peel was boiled in hot water for 1 h until it was soft. It was allowed to cool down for 30 min before it was strained to separate the peelings from the solution. Lime (Ca(OH)2 ) was added to reach pH 11 and the mixture was provided a 30 min precipitation period before the pectin was strained by using a piece of cloth. Hydrochloric acid was further added to the solution to reach pH 4 and chilled overnight (10 h) for precipitation. The hesperetin or naringenin was extracted from the solution by sieving, placed on trays and oven dried at 50 ◦ C for 24 h. The extracted hesperetin or naringenin was later ground before it was used. 2.3. Determination of the hesperetin and naringenin concentration Hesperetin and naringenin concentrations were determined with reference to the methods of Bronner and Beecher (1995) and Kanaze et al. (2004), and analyzed by HPLC (Hitachi L-2130, Japan). Hesperetin and naringenin standards (H4125 and N-5893, Sigma Chem Co., 95% purity) were dissolved in methanol:water (1:1). The sample and standard were added to 100 L 1 M sodium acetate buffer (pH 5) and 40 L B-glucuronidase/sulphatase, and allowed to stand for 18 h at 37 ◦ C for hydrolysis. 2 mL ethanol was added, and filtering was accomplished with a 0.45 m filter. It was then dried and stored at −20 ◦ C. For analysis, 100 L of the mobile phase (methanol/water/acetic acid (40:58:2) (pH 7.1) was added to the samples and mixed for 30 s. A C18 steel column (250 mm × 4.6 mm, Lot: 6957) (Hitach, Japan) was used. Preconditioning involved 6 mL methanol and 6 mL 0.01 M HCl, 5 mL 10% methanol in 0.01 M HCl mixed with 3.0 mL 0.01 M HCl, and then washed with 3.0 mL 0.01 M HCl. The machine flow rate was set at 1 mL/min, UV detector scanned at 280 nm, injected sample was 20 L, and mobile phase was methanol/water/acetic acid (40:58:2). Hesperetin and naringenin were run concurrently as standards, and displayed over 95% recovery. 2.4. Determination of egg weight, yolk weight, shell thickness and shell strength Eggs and egg yolk were weighed using an electronic balance. Egg shell strength was evaluated using a press meter (FHK, Japan) to gauge the force producing cracking under longitudinal compression, and the shell maximum deformation was recorded (g/cm2 ). The average shell thickness was calculated from each of its two ends and a middle section of egg shell measured with a micrometer. 2.5. Determination of yolk cholesterol Yolk cholesterol was determined following the method described by Pasin et al. (1998): 3 g of egg yolk was diluted with 27 mL NaCl solution (20 g/kg), stirred for 2 h by using a magnetic stirrer, then 1 mL (egg yolk and NaCl solution) was further diluted with 9 mL NaCl solution (20 g/kg). A cholesterol reagent kit (Cat. No., 11491458, Roche Cholesterol assay) was used for enzymatic determination using cholesterol esterase and cholesterol oxidase. The color intensity was determined photometrically at 540 nm, using a spectrophotometer (Hitachi U-2000, Japan). A known concentration of yolk cholesterol (Elkin, 2009) was confirmed by this method. The total cholesterol/egg was calculated as yolk cholesterol times yolk weight. 2.6. Determination of serum traits Serum cholesterol and triglyceride (TG) were determined by commercial enzymatic test kits (Cat. No. 11730711, Roche/Hitachi), and were determined photometrically using a serum biochemical auto-analyzer (Roche, Co., Switzerland). 2.7. Antioxidant activity assay Serum superoxide dismutase (SOD) and catalase activities were determined according to the method suggested by Ellerby and Bredesen (2000), with SOD and catalase (S-8160 and C-1345, Sigma Co., USA) used as standards. Glutathione peroxidase (GSH-Px) activity was conducted following the method reported by Bhat et al. (1992). Scavenging superoxide activity was determined following the method described by Shi and Dalal (1991). A cuvette contained 0.90 mL of reaction buffer (including 50 mM potassium phosphate, pH 7.8; 1 mM EDTA; 100 M nitroblue tetrazolium (NDT); 0.25 g/kg triton X-100; 100 M hypoxanthine) and 50 L sample; the reaction began after 0.025 U/ML xanthine oxidase was added. The measurement was taken by using a spectrophotometer at 560 nm for 5 min. Trolox equivalent antioxidant capacity (total antioxidant capacity) measurement followed Erel’s (2004) procedure. Solution 1 contained 0.4 M acetate buffer solution, 0.4 M sodium acetate (CH3 COONa), 0.4 M glacial acetic acid, and was adjusted to pH 5.8. Solution 2 containing 30 mM acetate buffer solution, 30 mM sodium acetate (CH3 COONa), 30 mM glacial acetic
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Table 2 Effect of hesperetin supplementation on egg production and quality in laying hens. Variables
Egg production (%) Eggshell strength (g/cm2 ) Eggshell thickness (mm) Egg weight (g) Yolk weight (g) Yolk weight/egg weight Cholesterol/g yolk (mg/g) Total cholesterol/egg (mg/g)
Hesperetin (g/kg)
SEM
0
0.5
1
2
4
95.8a 3.13 0.45 55 16.3 0.29 11.8a 191a
94.9ab 3.15 0.46 56 16.4 0.29 11.2ab 184ab
94.5bc 3.15 0.47 54 16.6 0.29 10.4bc 172bc
95.4ab 3.14 0.47 54 16.0 0.29 9.9c 158c
92.4c 3.25 0.46 55 15.9 0.28 10.8abc 172bc
1.1 0.2 0.02 0.9 0.3 0.02 0.1 1.4
P-value Linear
Quadratic
0.12 0.09 0.28 0.64 0.80 0.38 0.14 0.13
<0.01 0.20 0.21 0.69 0.84 0.88 <0.01 <0.01
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05).
acid, 2.86 M H2 O2 , and was adjusted to pH 6.0. Then, 0.549 g ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) was dissolved in 100 mL of prepared solution 2. A volume of 200 L of solution 1 was added to 5 L serum sample and mixed with 20 L solution 2. The mixture was allowed to stand for 5 min and then scanned at wavelength 660 nm using a spectrophotometer. Trolox (a derivate of Vitamin E) was used as the standard. Iron-chelating ability was determined following the procedure described by Osawa et al. (1992). The chemicals (0.002 mM linoleic acid; 0.05 M trizma; 0.5% Tween-20) were prepared on a per sample basis. After being mixed thoroughly and adjusted to pH 7.4, 0.0001 M ferrous sulphate (FeSO4 ), 0.0015 M H2 O2 and 25 L sample were added. The mixture was scanned at 234 nm by using a spectrophotometer. The ABS value was recorded at 0 h and after 15 h at a temperature of 25 ◦ C. Calculation = (1 − (ABS/control)×100 using water as the control. 2.8. Statistical analysis The effect of hesperetin and naringenin treatment on different parameters was evaluated by ANOVA using the GLM procedure of SAS, and variances between groups were analyzed with SAS software (SAS Institute and Inc., 1998). Differences between means were determined using Duncan’s new Multiple Range Test, according to the following model: Y = + Ti + eij where Y denotes the dependent variable, denotes the mean, T is the treatment effect and e is random residual error term. All values were presented as means and SEM; the level of ‘significantly different’ was set at P50.05. The linear and quadratic responses were determined with contrasts among the means (SAS Institute and Inc., 1998). 3. Results 3.1. Identification and quantification of hesperetin and naringenin The closely matched retention times confirmed hesperetin and naringenin to be main components in orange and grapefruit peels, respectively. The average concentration of hesperetin and naringenin used in these experiments were 350 g/kg and 357 g/kg, respectively. The other main component was lime, with a content of approximately 3.25, 6.5, 13.0, 26.0 g/kg lime in the 0.5, 1, 2, 4 g/kg hesperetin- and naringenin-supplemented groups, respectively. 3.2. Effect of hesperetin and naringenin on egg production Egg production was lowered (P<0.05) in the 4 g/kg hesperetin-supplemented group, as compared with the control group (Table 2). However, there were no differences in the egg weight, yolk weight and yolk weight/egg weight (P>0.05). Egg production was higher (P<0.05) in the 1 g/kg naringenin-supplemented group, as compared with the control group (Table 3). However, yolk weight in the 2 g/kg group was higher (P<0.05) than in the control group. There was no difference in the egg weight and yolk weight/egg weight (P>0.05). 3.3. Effect of hesperetin and naringenin on egg quality There were no significant differences in eggshell strength and thickness in both hesperetin- and naringenin-supplemented groups (Tables 2 and 3). Cholesterol/g yolk and total cholesterol/egg in hesperetin-supplemented group showed a quadratic (P<0.01) dose response, and was reduced (P<0.05) in the 1 and 2 g/kg supplemented group, as compared to the control group (Table 2). Cholesterol/g yolk and total cholesterol/egg were reduced (P<0.05) in the hesperetin-supplemented groups by 8–18%, as compared with the control group.
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Table 3 Effect of naringenin supplementation on egg production and quality in laying hens. Variables
Naringenin (g/kg)
Egg production (%) Eggshell strength (g/cm2 ) Eggshell thickness (mm) Egg weight (g) Yolk weight (g) Yolk weight/egg weight Cholesterol/g yolk (mg/g) Total cholesterol/egg (mg/g)
SEM
0
0.5
1
2
4
94.6bc 3.11 0.45 56 16.3b 0.29 11.8a 192a
95.9ab 3.13 0.46 59 16.2b 0.30 10.7b 173b
96.3a 3.14 0.47 55 16.5ab 0.30 10.9b 181b
95.8ab 3.14 0.47 58 17.4a 0.30 9.1c 158c
93.3c 3.13 0.46 56 16.5ab 0.29 10.8b 178b
1.1 0.1 0.02 1.1 0.5 0.1 0.1 2.4
P value Linear
Quadratic
0.14 0.11 0.29 0.68 0.15 0.16 0.11 0.12
<0.01 0.21 0.21 0.29 <0.01 0.15 <0.01 <0.01
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). Table 4 Effect of hesperetin and naringenin supplementation on the serum traits in laying hens. Variables
0 g/kg
0.5 g/kg
1 g/kg
2 g/kg
4 g/kg
SEM
P value Linear
Quadratic
Hesperetin Cholesterol (mg/dl) Triglyceride (mg/dl)
176ab 1647ab
151ab 1538ab
137bc 1496ab
110c 1160b
116c 1236b
25.3 207.6
0.07 0.44
0.04 0.03
Naringenin Cholesterol (mg/dl) Triglyceride (mg/dl)
169a 1990a
111c 1529ab
150ab 1629ab
121bc 1133b
141abc 1603ab
23.1 305.4
0.22 0.25
<0.01 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05).
Furthermore, results indicated that naringenin supplementation at 2 g/kg was sufficient to exert a significant hypocholesterolemic effect on egg yolk in laying hens (P<0.05) (Table 3). The cholesterol/g yolk and total cholesterol/egg in naringenin-supplemented group was also reduced (P<0.05) at 12–22%, and showed a quadratic dose response. 3.4. Effect of hesperetin and naringenin on serum traits Results from both experiments showed that the supplementation of hesperetin at 2 and 4 g/kg, and naringenin at 0.5 and 2 g/kg were significantly lowered (P<0.05) the serum total cholesterol concentration than the control group. The supplementation of hesperetin at 2 and 4 g/kg, and naringenin at 2 g/kg were significantly lowered (P<0.05) the serum triglyceride concentration as compared to the control group (Table 4). These groups also displayed a quadratic dose response. 3.5. Effect of hesperetin and naringenin on antioxidant activities Comparing the antioxidant enzyme activities with the control group, SOD, catalase, scavenging O2 − and iron-chelating ability were higher (P<0.05) in the 2 g/kg hesperetin-supplemented group, while the 2 g/kg naringenin-supplemented group increased (P<0.05) SOD, catalase, trolox equivalent antioxidant capacity, scavenging O2 − and iron-chelating ability. Hesperetin and naringenin supplementation at 0.5 g/kg level was significant differences in SOD activity, and at 2 g/kg level was the highest. Most of them showed quadratic dose responses, except for iron-chelating in hesperetin and trolox equivalent antioxidant capacity in naringenin, which showed a linear dose response. There were no differences in the GSH Px at all levels in both naringenin- and hesperetin-supplemented groups (Tables 5 and 6). 4. Discussion 4.1. Effects of hesperetin and naringenin on egg quality and production The results of this study showed the potential of hesperetin and naringenin to lower egg yolk cholesterol content. Lien et al. (2008) also reported reduced egg yolk cholesterol content when adding 0.5 g/kg of hesperetin and naringenin to the layers’ diet. Egg yolk cholesterol may be reduced due to the inhibition of HMG-CoA reductase activity, the key enzyme that regulates the rate of cholesterol biosynthesis in the liver, thus inhibiting the enzyme can reduce cholesterol synthesis (Lee et al., 1999, 2003; Bok et al., 2000; Kim et al., 2003). The cholesterol content of the yolk is primarily dependent on the cholesterol content of vitellogenin and triglyceride-rich lipoproteins-VLDL. Virtually all of the cholesterol found in the egg yolk is originated in the liver, where it is synthesized, and transported in the plasma to the ovaries where they are taken up into the developing follicles by receptor-mediated endocytosis.
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Table 5 Effect of hesperetin supplementation on anti-oxidant activity in laying hens. Variables
SOD (n mol/mg protein) Catalase (n mol/mg protein) GSH Px (n mol/mg protein) Trolox equivalent antioxidant capacity (mM/l) Iron-chelating (%) Scavenging O2 − (%)
Hesperetin (g/kg)
SEM
0
0.5
1
2
4
23.5c 73.3b 20.0 38.1 76.1b 84.4b
41.5ab 81.8a b 21.6 38.7 80.1ab 86.6ab
42.6a 86.7a b 20.9 37.6 87.1a 88.3ab
43.7a 88.6a 21.8 36.3 86.8a 94.3a
34.5ab 86.5a b 21.1 33.7 87.1a 86.5ab
8.4 6.2 0.7 2.1 5.1 3.7
P value Linear
Quadratic
0.28 0.09 0.08 0.08 0.02 0.11
0.04 0.02 0.16 0.12 0.10 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). SOD: superoxide dismutase. GSH Px: glutathione peroxidase. Table 6 Effect of naringenin supplementation on anti-oxidant activity in laying hens. Variables
SOD (n mol/mg protein) Catalase (n mol/mg protein) GSH Px (n mol/mg protein) Trolox equivalent antioxidant capacity (mM/l) Iron-chelating (%) Scavenging O2 - (%)
Naringenin (g/kg)
SEM
0
0.5
1
2
4
24.7c 78.3b 20.3 34.2b 77.9b 84.5b
37.7b 83.1ab 21.8 45.9ab 83.4ab 87.5ab
43.2ab 89.4ab 22.0 47.5ab 81.9ab 87.2ab
46.6a 91.9a 22.6 53.9a 87.7a 94.2a
40.1ab 83.0ab 22.5 54.5a 86.0ab 86.4ab
2.6 4.7 0.9 8.6 3.8 2.8
P value Linear
Quadratic
0.12 0.08 0.11 0.04 0.07 0.08
0.02 0.01 0.19 0.16 <0.01 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). SOD: superoxide dismutase. GSH Px: glutathione peroxidase.
The maximum reduction of egg yolk cholesterol by hesperetin and naringenin was 18% and 22%, respectively, although the percentage reduction was less than those reported for triparanol (77%), azasterols (80%) and atorvastatin (46%) (Dam et al., 1979; Cecil et al., 1981; Elkin et al., 1999). However, triparanol and azasterols had serious side effects; and atorvastatin largely reduced egg production (Elkin, 2006, 2007). Hesperetin and naringenin are natural organic flavonoids compounds and displayed antioxidant capacity in this study, and in other reports (Jeon et al., 2001; Seo et al., 2003; Kim et al., 2004). Some investigations compared hesperetin and naringenin with lovastatin – a cholesterol reducing medicine, and found that they had comparable effects (Kim et al., 2003). Thus, the use of hesperetin and naringenin to produce low cholesterol egg production may more acceptable to consumers. Furthermore, the extractions of hesperitin and naringenin from orange and grapefruit peels are simple and inexpensive. There were no effects of hesperetin and naringenin on eggshell strength and eggshell thickness. The level of lime (26 g/kg) present in the hesperetin and naringenin could have influenced these measurements as lime has been shown previously to influence eggshell quality (Plimmer and Lowndes, 1924). However this did not occur in our study, and furthermore, there are no reports showing that lime content in the diet influences the serum and yolk cholesterol contents or antioxidant capacity. 4.2. Effects of hesperetin and naringenin on serum traits This study has demonstrated that both hesperetin and naringenin supplementation (2 g/kg) could significantly lower the serum cholesterol concentration, corresponding to other findings (Lee et al., 2003; Seo et al., 2003; Kim et al., 2004; Lien et al., 2008). Similarly, serum triglyceride was significantly lowered in 2 g/kg hesperitin and naringenin supplementation. This finding is consistent with Lee et al. (1999, 2003), Bok et al. (2000), and Lien et al. (2008). The reduction of serum cholesterol could be due to the inhibition of the key enzymes in the cholesterol synthesis, HMG-CoA reductase. The blockage of cholesterol synthesis results in a lower intracellular supply of cholesterol. Wilcox et al. (1998) and Borradaile et al. (1999) reported that hesperetin and naringenin not only decreased cholesterol synthesis but also inhibited ACAT activity in HepG2 cells. Inhibition of ACAT reduced the hepatic production of apo-B containing lipoproteins (Burnett et al., 1998) such as VLDL, thereby reducing the plasma triglyceride concentration. 4.3. Effects on antioxidant activities This study showed that SOD level was relatively high for hesperetin and naringenin at all supplementation levels; even a low level (0.5 g/kg) of supplementation was able to exert high serum SOD activity. The results correspond with a similar study on the supplementation of hesperetin and naringenin by Lien et al. (2008) on layer hens. Furthermore, Jeon et al. (2001), Seo et al. (2003) and Kim et al. (2004) also reported a high level of hepatic SOD activity in naringenin-fed rabbits,
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rats and mice. Catalase activity was increased at 2 g/kg supplementation level for both hesperetin and naringenin. Jeon et al. (2001) and Kim et al. (2004) also found that catalase activity was increased in rabbits and mice fed with 0.5 g/kg or 0.2 g/kg naringenin. The increased SOD activity by hesperetin and naringenin supplementation resulted in a reduced superoxide anion level. GSH-Px activity remained low, probably due to higher SOD activity that might have led to a reduced peroxide level in the serum. Trolox equivalent antioxidant capacity showed no difference in all hesperetin supplementation levels while higher activity was detected at 2 and 4 g/kg naringenin supplementation. Scavenging O2 − activity was higher at both 2 g/kg of hesperetin- and naringenin-supplemented groups, which is consistent with the study of Chen et al. (1990). Lien et al. (2008) also found that hesperetin and naringenin have a strong superoxide free radical scavenging activity. The results suggested that hesperetin and naringenin can terminate chain radical reaction by donating hydrogen atoms to the peroxy hydroxyl (Cook and Samman, 1996). Since both hesperetin and naringenin are rich in OH groups to supply hydrogen atoms for free radicals to block the oxidation chain reaction, both hesperetin and naringenin have antioxidant activity. Egg production was reduced in the 4 g/kg hesperetin-supplemented group, indicating that the supplementation level of 4 g/kg is too high; but the reason remains unknown. According to the results on yolk cholesterol, serum traits and antioxidant activity, the supplemental levels in most results displayed a quadratic dose response and indicated that the supplementation level of 2 g/kg for both hesperetin and naringenin seemed to be the optimum level for reducing serum and yolk cholesterols contents and enhancing antioxidant capacity. 5. Conclusion Data from both experiments suggests that both hesperetin and naringenin can lower plasma and egg yolk cholesterol levels and improve the antioxidant capacity. Our data suggests that the optimum level of supplementation for both hesperetin and naringenin is 2 g/kg in the basal diet for a reduction in serum and yolk cholesterols contents and enhanced antioxidant capacity, without impairment of egg production. References Bhat, G.B., Tinsley, S.B., Tolson, J.K., Patel, J.M., 1992. Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury. J. Cell. Physiol. 151, 228–238. Bok, S.H., Shin, Y.W., Bae, K.H., Jeong, T.S., Kwon, Y.K., Park, Y.B., Choi, M.S., 2000. Effects of Naringenin and Lovastatin on plasma and hepatic lipids in high fat and high-cholesterol fed rat. Nutr. Res. 20, 1007–1015. Borradaile, N.M., Carroll, K.K., Kurowska, E.M., 1999. Regulation of hepG2 cell apolipoprotein B metabolism by citrus flavanones hesperetin and naringenin. Lipids 34, 591–598. Bronner, W.E., Beecher, G.R., 1995. Extraction and measurement of prominent flavonoids in orange and grapefruit juice concentrates. J. Chromatogr. 705, 247–256. Burnett, J.R., Wilcox, L.J., Telford, D.E., Kleinstiver, S.J., Barrett, P.H.R., Huff, M.W., 1998. Inhibition of cholesterol esterification by Dup 128 decreases hepatic apolipoprotein B secretion in vivo: effect of dietary fat and cholesterol. Biochim. Biophys. Acta. 1393, 63–79. Cecil, H.C., Bitman, J., Svoboda, J.A., Thompson, M.J., 1981. Effects of branched and straight chain amines and azasteroids on blood and egg cholesterol of White Leghorn chickens. Poult. Sci. 60, 795–804. Cook, N.C., Samman, S., 1996. Flavonoids – chemistry, metabolism, cardioprotective effects, and dietary sources. J. Nutr. Biochem. 7, 66–67. Chen, Y.T., Zheng, R.L., Jia, Z.J., Ju, Y., 1990. Flavonoids as superoxide scavengers and antioxidants. Free Radic. Biol. Med. 9, 19–21. Dam, R., Labate, M.E., Tam, S.W., Cuervo-Torres, C., 1979. Effects of diazacholesterol, triparanol, and -sitosterol on egg cholesterol deposition in Coturnix quail. Poult. Sci. 58, 985–987. Ellerby, L.M., Bredesen, D.E., 2000. Measurement of cellular oxidation, reactive oxygen species, and antioxidant enzymes during apotosis. Methods Ezymol. 322, 413–421. Erel, O., 2004. A novel automated direct measurement method for total antioxidant capacity using new generation, more stable ABTS radical cation. Clin. Biochem. 37, 277–285. Elkin, R.G., Yan, Z., Zhong, Y., Donkin, S.S., Buhman, K.K., Story, J.A., Turek, J.J., Porter, R.E., Anderson, M., Homan, R., Newton, R.S., 1999. Select 3-hydroxy-3methylglutaryl-coenzyme A reductase inhibitors vary in their ability to reduce egg yolk cholesterol levels in laying hens through alteration of hepatic cholesterol biosynthesis and plasma VLDL composition. J. Nutr. 129, 1010–1019. Elkin, R.G., 2006. Reducing shell egg cholesterol content. I. Overview, genetic approaches, and nutritional strategies. World Poult. Sci. J. 62, 665–687. Elkin, R.G., 2007. Reducing shell egg cholesterol content. II. Review of approaches utilizing non-nutritive dietary factors or pharmacological agents and an examination of emerging strategies. World Poult. Sci. J. 63, 5–30. Elkin, R.G., 2009. Additional perspectives on analytical techniques and standardization: cholesterol and fatty acid contents of eggs, and organs. Poult. Sci. 88, 249–250. El-Nawawi, S.A., 1995. Extraction of citrus glucosides. Carbohydr. Polym. 27, 1–4. Erlund, I., Meririnne, E., Alfthan, G., Aro, A., 2001. Plasma kinetics and urinary extraction of flavanones naringenin and hesperetin in humans after ingestion of orange juice and grapefruit juice. J. Nutr. 131, 235–241. Friedman, M., 2007. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol. Nutr. Food Res. 51, 116–134. Jeon, S.M., Bok, S.H., Lee, M.K., Nam, K.T., Park, Y.B., Rhee, S.J., Chio, M.S., 2001. Antioxidative activity of naringenin and lovastatin in high cholesterol-fed rabbits. Life Sci. 69, 2855–2866. Kanaze, F.I., Kokkalou, E., Georgarakis, M., Niopas, I., 2004. Validated high-performance liquid chromatographic method utilizing solid-phase extraction for the simultaneous determination of naringenin and hesperetin in human plasma. J. Chromatogr. B 801, 363–367. Kim, H.K., Jeong, T.S., Lee, M.K., Park, Y.B., Choi, M.S., 2003. Lipid-lowering efficacy of hesperetin metabolites in high-cholesterol fed rats. Clin. Chim. Acta. 327, 129–137. Kim, H.J., Oh, G.T., Park, Y.B., Lee, M.K., Seo, H.J., Choi, M.S., 2004. Naringin alters the cholesterol biosynthesis and antioxidant enzyme activities in LDL receptor-knockout mice under cholesterol fed condition. Life Sci. 74, 1621–1634. Krauss, R.M., Eckel, R.H., Howard, B., Appel, L.J., Daniels, S.R., Deckelbaum, R.J., Erdman, J.W., Kris-Etherton, P., Goldberg, I.J., Kotchen, T.A., Lichtensten, A.H., Mitch, W.E., Mullis, R., Robinson, K., Wylie-Rosett, J., Jeor, S.S., Suttie, J., Tribble, D.L., Bazzarre, T.L., 2000. AHA dietary guidelines. Revision 2000: a statement for healthcare professionals from the nutrition committee of the American Heart Association. Stroke 31, 2751–2766.
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Kurowska, E.M., Borradaile, N.M., Spence, J.D., Carroll, K.K., 1999. Hypocholesterolemic effects of dietary citrus juices in rabbits. Nutr. Res. 20, 121–129. Lee, S.H., Jeong, T.S., Park, Y.B., Kwon, Y.K., Choi, M.S., Bok, S.H., 1999. Hypocholesterolemic effect of hesperetin mediated by inhibition of 3-hydroxy 3-methylgultaryl coenzyme a reductase and acyl coenzyme A: cholesterol acyltransferase in rats fed high-cholesterol diet. Nutr. Res. 19, 1245–1258. Lee, M.K., Moon, S.S., Lee, S.E., Bok, S.H., Jeong, T.S., Park, Y.B., Choi, M.S., 2003. Naringenin 7-o-cetyl ether as inhibitor of HMG-CoA reductase and modulator of plasma and hepatic lipids in high cholesterol-fed rats. Bioorg. Med. Chem. 11, 393–398. Lien, T.F., Yeh, H.S., Su, W.T., 2008. Effect of adding extracted hesperetin, naringenin and pectin on egg cholesterol, serum traits and antioxidant activity in laying hens. Arch. Anim. Nutr. 62, 33–43. National Cholesterol Education Program, 2002. Third Report of The Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, National Institutes of Health (NIH) Publication No. 02-5215. National Heart, Lung, and Blood Institute, Bethesda, MD, pp. 284. Osawa, T., Katsuzaki, H., Hagiwara, Y., Hagiwara, H., Shibamoto, T., 1992. A novel antioxidant isolated from young green barley leaves. J. Agric. Food Chem. 40, 1135–1141. Pasin, G., Smith, G.M., Mahony, M.O., 1998. Rapid determination of total cholesterol in egg yolk using commercial diagnostic cholesterol reagent. Food Chem. 61, 255–259. Plimmer, R.H.A., Lowndes, J., 1924. The changes in the lime content of the hen’s egg during development. Biochem. J. 18, 1163–1169. SAS Institute Inc., 1998. SAS User Guide: Statistic. SAS Inst., Cary, NC. Seo, H.J., Jeong, K.S., Lee, M.K., Park, Y.B., Jung, U.J., Kim, H.J., Choi, M.S., 2003. Role of naringenin supplement in regulation of lipid and ethanol metabolism in rats. Life Sci. 73, 933–946. Shi, X., Dalal, N.S., 1991. Antioxidant behavior of caffeine: effect scavenging of hydroxyl radicals. Food Chem. Toxicol. 29, 1–6. Wilcox, L.J., Borradaile, N., Kurowska, E., Telford, D.E., Huff, M.W., 1998. Naringenin, a citrus flavonoid, markedly decreases apoB secretion in hepG2 cells and inhibits acyl CoA: cholesterol acyl-transferase. Circulation 98, 537 (abstract). Yang, C.S., Sang, S., Lambert, J.D., Lee, M.J., 2008. Bioavailability issues in studying the health effects of plant polyphenolic compounds. Mol. Nutr. Food Res. 52, S139–S151.
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Effects of supplemental levels of hesperetin and naringenin on egg quality, serum traits and antioxidant activity of laying hens S.Ting, H.S. Yeh, T.F. Lien ∗ Department of Animal Science, National Chiayi University, 300 University Road, Luh Liau Li, Chiayi 600, Taiwan, ROC
a r t i c l e
i n f o
Article history: Received 10 July 2009 Received in revised form 30 September 2010 Accepted 4 October 2010
Keywords: Hesperetin Naringenin Egg quality Serum traits Antioxidant activity Laying hens
a b s t r a c t Hesperetin and naringenin phytochemicals are naturally occurring flavanoids in citrus fruits. The purpose of this study was to evaluate the effects of supplementing different levels of extracted hesperetin and naringenin on egg quality, serum traits and antioxidant activity in laying hens. Two experiments were conducted, each for 10 weeks, in a completely randomized experiment design. Each had 100 Leghorn laying hens (26 weeks old) randomly assigned into five groups (n = 20) based on dietary categories of hesperetin 0, 0.5, 1, 2, 4 g/kg and naringenin 0, 0.5, 1, 2, 4 g/kg. Experimental results indicated that there was increased (P<0.05) egg production in the 1 g/kg naringenin-supplemented group, but lower (P<0.05) egg production in the hesperetin- and naringenin-supplemented groups given 4 g/kg. Cholesterol content (per gram yolk) and total cholesterol content (per egg) were lower (P<0.05) in the hesperetin- and naringenin-supplemented groups as compared to the control group, and the 2 g/kg hesperetin- and naringenin-supplemented groups showed the most significant difference. Both serum cholesterol and triglyceride concentrations were lower (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups. The SOD and catalase activities, scavenging O2 − and iron-chelating abilities were higher (P<0.05) in the 2 g/kg hesperetin- and naringenin-supplemented groups, and the trolox equivalent antioxidant capacity was higher (P<0.05) in the 2 g/kg naringenin-supplemented group. The results confirmed that both hesperetin and naringenin could lower serum and egg yolk cholesterol levels, and improve the antioxidant activities, however the measured variables generally showed significant quadratic responses to increasing amounts of the compounds. The recommended supplementation level of hesperetin and naringenin is 2 g/kg of the basal diet for reduced serum and yolk cholesterols contents and increased antioxidant capacity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Eggs have a high biological value and contain a wide array of nutrients needed by the human body to maintain good health. Eggs have been shown to be a major source of dietary cholesterol (180–250 mg) (Elkin, 2009). However, dietary cholesterol significantly elevates plasma cholesterol in about 15–30% of the population (hyper-responders) (Elkin, 2006). Thus, some organizations, such as the American Heart Association and the National Cholesterol Education Program, recommend a population wide limitation of dietary cholesterol intake to less than 300 mg/day (Krauss et al., 2000; National Cholesterol Education Program, 2002). One of the dietary recommendations in the prevention of coronary heart disease for example is to limit egg consumption. Thus, demand for a lower cholesterol concentration has led to many attempts to reduce
∗ Corresponding author. Tel.: +886 5 2717536; fax: +886 5 2750134. E-mail address: tfl[email protected] (T.F. Lien). 0377-8401/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2010.10.001
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Table 1 Composition of basal diets (as feed basis). Ingredients
g/kg
Yellow corn meal Soybean meal, 445 g crude protein/kg Fish meal, 600 g crude protein/kg Soybean oil Dicalcium phosphate Limestone, pulverized Sodium chloride Vitamin premixa Mineral premixb Oyster shells dl-Methionine Choline chloride, 50 g/kg
607.5 240.0 30.0 20.0 13.0 42.5 3.0 0.5 2.0 40.0 0.5 1.0
Total
1000.0
Calculated value: Crude protein (g/kg) ME (MJ/kg) Calcium (g/kg) Available phosphorous (g/kg) Lysine (g/kg) Methionine + cystine (g/kg)
179 11.8 34.0 4.7 9.8 6.2
Analyzed value: Hesperitin (g/kg) Naringenin (g/kg)
Trace Trace
a Vitamin premix supplied the following per kilogram of diet: Vitamin A, 12,500 IU; Vitamin D3 , 3125 ICU; Vitamin E, 37.5 IU; Vitamin K3 , 6.25 mg; Vitamin B1 , 3.75 mg; Vitamin B2 , 12.5 mg; Vitamin B6 , 10.0 mg; Pantothenate, 18.8 mg; Niacin, 50 mg; Biotin, 0.06 mg; Folic acid, 1.25 mg; Vitamin B12 , 0.05 mg. b Mineral premix supplied the following per kilogram of diet: Cu (CuSO4 ·5H2 O, 25.45% Cu) 6 mg; Fe (FeSO4 ·7H2 O, 20.29% Fe) 50 mg; Mn (MnSO4 ·H2 O, 32.49% Mn) 40 mg; Zn (ZnO, 80.35% Zn) 60 mg; Se (NaSeO3 , 45.56% Se) 0.075 mg.
the cholesterol concentration in eggs. This has involved a number of different approaches, including genetic selection, avian transgenesis, nutritional interventions such as n-3 fatty acids (Elkin, 2006), non-nutritive dietary factors such as oxysterol (Elkin, 2007), and (or) pharmacological manipulation (Elkin, 2007). Hesperetin and naringenin and are naturally occurring flavonoids that can be extracted from the peels of oranges (Citrus sinensis) and grapefruits (Citrus × Paradisi). Flavonoids have exhibited a variety of biological and pharmacological activities, including the inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and Acyl-CoA: cholesterol acyltransferase (ACAT) enzymes (Bok et al., 2000; Kim et al., 2004), free radical scavenging, anti-inflammation, and inhibition of tumor promotion (Friedman, 2007; Yang et al., 2008; Seo et al., 2003). The principal citrus flavonoids, hesperetin from oranges and naringenin from grapefruits are structurally similar to genistein which has been tested as a potential agent for improving the cholesterol metabolism in diet-induced hypercholesterolemic animals (Kurowska et al., 1999). Hesperetin and naringenin have also been found to exhibit estrogenic, anti-carcinogenic (Erlund et al., 2001), and strong antioxidant functions (Jeon et al., 2001). A previous study by Lien et al. (2008) indicated that adding hesperetin and naringenin at 0.5 g/kg lowered serum and yolk cholesterol, and improved antioxidant ability. However, the work of Lien et al. (2008) only used an inclusion level of 0.5 g/kg and it is possible that the optimum inclusion level is above this amount. Therefore, the aim of the present study was to investigate the effects of supplementing graded levels of hesperetin or naringenin on egg quality, serum traits and antioxidant activity of laying hens, and assess whether these responses behave in a linear or quadratic manner. 2. Materials and methods 2.1. Experimental design and measurements Two experiments were conducted simultaneously; each used 100 individually-caged, single-comb white Leghorn laying hens (26 weeks old) randomly assigned into 5 groups (n = 20) and fed with diets containing hesperetin at 0, 0.5, 1, 2, 4 g/kg or naringenin at 0, 0.5, 1, 2, 4 g/kg. Hens were each fed with about 110 g diet every day, water was supplied ad libitum and lighting was controlled (15 h) throughout the 10-week experimental period. Eggs were collected daily and egg production was recorded. Four eggs per hen per treatment were collected during the end of the experiment. Blood (about 10 mL) was collected (from brachial wing vein) in a glass tube (16 mm × 100 mm) at the end of the experiment, blood samples were left to stand at room temperature for clotting, and then the serum was obtained by centrifugation at 1500 × g for 10 min. The composition of the basal diets for both experiments is shown in Table 1. This study was approved by the Animal Care and
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Use Committee of the University. Animals used in this experiment were cared for under the guidelines stated in the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. 2.2. Hesperetin and naringenin extraction method The hesperetin and naringenin extraction from peels followed the method described by El-Nawawi (1995). Orange peel or grapefruit peel was boiled in hot water for 1 h until it was soft. It was allowed to cool down for 30 min before it was strained to separate the peelings from the solution. Lime (Ca(OH)2 ) was added to reach pH 11 and the mixture was provided a 30 min precipitation period before the pectin was strained by using a piece of cloth. Hydrochloric acid was further added to the solution to reach pH 4 and chilled overnight (10 h) for precipitation. The hesperetin or naringenin was extracted from the solution by sieving, placed on trays and oven dried at 50 ◦ C for 24 h. The extracted hesperetin or naringenin was later ground before it was used. 2.3. Determination of the hesperetin and naringenin concentration Hesperetin and naringenin concentrations were determined with reference to the methods of Bronner and Beecher (1995) and Kanaze et al. (2004), and analyzed by HPLC (Hitachi L-2130, Japan). Hesperetin and naringenin standards (H4125 and N-5893, Sigma Chem Co., 95% purity) were dissolved in methanol:water (1:1). The sample and standard were added to 100 L 1 M sodium acetate buffer (pH 5) and 40 L B-glucuronidase/sulphatase, and allowed to stand for 18 h at 37 ◦ C for hydrolysis. 2 mL ethanol was added, and filtering was accomplished with a 0.45 m filter. It was then dried and stored at −20 ◦ C. For analysis, 100 L of the mobile phase (methanol/water/acetic acid (40:58:2) (pH 7.1) was added to the samples and mixed for 30 s. A C18 steel column (250 mm × 4.6 mm, Lot: 6957) (Hitach, Japan) was used. Preconditioning involved 6 mL methanol and 6 mL 0.01 M HCl, 5 mL 10% methanol in 0.01 M HCl mixed with 3.0 mL 0.01 M HCl, and then washed with 3.0 mL 0.01 M HCl. The machine flow rate was set at 1 mL/min, UV detector scanned at 280 nm, injected sample was 20 L, and mobile phase was methanol/water/acetic acid (40:58:2). Hesperetin and naringenin were run concurrently as standards, and displayed over 95% recovery. 2.4. Determination of egg weight, yolk weight, shell thickness and shell strength Eggs and egg yolk were weighed using an electronic balance. Egg shell strength was evaluated using a press meter (FHK, Japan) to gauge the force producing cracking under longitudinal compression, and the shell maximum deformation was recorded (g/cm2 ). The average shell thickness was calculated from each of its two ends and a middle section of egg shell measured with a micrometer. 2.5. Determination of yolk cholesterol Yolk cholesterol was determined following the method described by Pasin et al. (1998): 3 g of egg yolk was diluted with 27 mL NaCl solution (20 g/kg), stirred for 2 h by using a magnetic stirrer, then 1 mL (egg yolk and NaCl solution) was further diluted with 9 mL NaCl solution (20 g/kg). A cholesterol reagent kit (Cat. No., 11491458, Roche Cholesterol assay) was used for enzymatic determination using cholesterol esterase and cholesterol oxidase. The color intensity was determined photometrically at 540 nm, using a spectrophotometer (Hitachi U-2000, Japan). A known concentration of yolk cholesterol (Elkin, 2009) was confirmed by this method. The total cholesterol/egg was calculated as yolk cholesterol times yolk weight. 2.6. Determination of serum traits Serum cholesterol and triglyceride (TG) were determined by commercial enzymatic test kits (Cat. No. 11730711, Roche/Hitachi), and were determined photometrically using a serum biochemical auto-analyzer (Roche, Co., Switzerland). 2.7. Antioxidant activity assay Serum superoxide dismutase (SOD) and catalase activities were determined according to the method suggested by Ellerby and Bredesen (2000), with SOD and catalase (S-8160 and C-1345, Sigma Co., USA) used as standards. Glutathione peroxidase (GSH-Px) activity was conducted following the method reported by Bhat et al. (1992). Scavenging superoxide activity was determined following the method described by Shi and Dalal (1991). A cuvette contained 0.90 mL of reaction buffer (including 50 mM potassium phosphate, pH 7.8; 1 mM EDTA; 100 M nitroblue tetrazolium (NDT); 0.25 g/kg triton X-100; 100 M hypoxanthine) and 50 L sample; the reaction began after 0.025 U/ML xanthine oxidase was added. The measurement was taken by using a spectrophotometer at 560 nm for 5 min. Trolox equivalent antioxidant capacity (total antioxidant capacity) measurement followed Erel’s (2004) procedure. Solution 1 contained 0.4 M acetate buffer solution, 0.4 M sodium acetate (CH3 COONa), 0.4 M glacial acetic acid, and was adjusted to pH 5.8. Solution 2 containing 30 mM acetate buffer solution, 30 mM sodium acetate (CH3 COONa), 30 mM glacial acetic
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Table 2 Effect of hesperetin supplementation on egg production and quality in laying hens. Variables
Egg production (%) Eggshell strength (g/cm2 ) Eggshell thickness (mm) Egg weight (g) Yolk weight (g) Yolk weight/egg weight Cholesterol/g yolk (mg/g) Total cholesterol/egg (mg/g)
Hesperetin (g/kg)
SEM
0
0.5
1
2
4
95.8a 3.13 0.45 55 16.3 0.29 11.8a 191a
94.9ab 3.15 0.46 56 16.4 0.29 11.2ab 184ab
94.5bc 3.15 0.47 54 16.6 0.29 10.4bc 172bc
95.4ab 3.14 0.47 54 16.0 0.29 9.9c 158c
92.4c 3.25 0.46 55 15.9 0.28 10.8abc 172bc
1.1 0.2 0.02 0.9 0.3 0.02 0.1 1.4
P-value Linear
Quadratic
0.12 0.09 0.28 0.64 0.80 0.38 0.14 0.13
<0.01 0.20 0.21 0.69 0.84 0.88 <0.01 <0.01
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05).
acid, 2.86 M H2 O2 , and was adjusted to pH 6.0. Then, 0.549 g ABTS (2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) was dissolved in 100 mL of prepared solution 2. A volume of 200 L of solution 1 was added to 5 L serum sample and mixed with 20 L solution 2. The mixture was allowed to stand for 5 min and then scanned at wavelength 660 nm using a spectrophotometer. Trolox (a derivate of Vitamin E) was used as the standard. Iron-chelating ability was determined following the procedure described by Osawa et al. (1992). The chemicals (0.002 mM linoleic acid; 0.05 M trizma; 0.5% Tween-20) were prepared on a per sample basis. After being mixed thoroughly and adjusted to pH 7.4, 0.0001 M ferrous sulphate (FeSO4 ), 0.0015 M H2 O2 and 25 L sample were added. The mixture was scanned at 234 nm by using a spectrophotometer. The ABS value was recorded at 0 h and after 15 h at a temperature of 25 ◦ C. Calculation = (1 − (ABS/control)×100 using water as the control. 2.8. Statistical analysis The effect of hesperetin and naringenin treatment on different parameters was evaluated by ANOVA using the GLM procedure of SAS, and variances between groups were analyzed with SAS software (SAS Institute and Inc., 1998). Differences between means were determined using Duncan’s new Multiple Range Test, according to the following model: Y = + Ti + eij where Y denotes the dependent variable, denotes the mean, T is the treatment effect and e is random residual error term. All values were presented as means and SEM; the level of ‘significantly different’ was set at P50.05. The linear and quadratic responses were determined with contrasts among the means (SAS Institute and Inc., 1998). 3. Results 3.1. Identification and quantification of hesperetin and naringenin The closely matched retention times confirmed hesperetin and naringenin to be main components in orange and grapefruit peels, respectively. The average concentration of hesperetin and naringenin used in these experiments were 350 g/kg and 357 g/kg, respectively. The other main component was lime, with a content of approximately 3.25, 6.5, 13.0, 26.0 g/kg lime in the 0.5, 1, 2, 4 g/kg hesperetin- and naringenin-supplemented groups, respectively. 3.2. Effect of hesperetin and naringenin on egg production Egg production was lowered (P<0.05) in the 4 g/kg hesperetin-supplemented group, as compared with the control group (Table 2). However, there were no differences in the egg weight, yolk weight and yolk weight/egg weight (P>0.05). Egg production was higher (P<0.05) in the 1 g/kg naringenin-supplemented group, as compared with the control group (Table 3). However, yolk weight in the 2 g/kg group was higher (P<0.05) than in the control group. There was no difference in the egg weight and yolk weight/egg weight (P>0.05). 3.3. Effect of hesperetin and naringenin on egg quality There were no significant differences in eggshell strength and thickness in both hesperetin- and naringenin-supplemented groups (Tables 2 and 3). Cholesterol/g yolk and total cholesterol/egg in hesperetin-supplemented group showed a quadratic (P<0.01) dose response, and was reduced (P<0.05) in the 1 and 2 g/kg supplemented group, as compared to the control group (Table 2). Cholesterol/g yolk and total cholesterol/egg were reduced (P<0.05) in the hesperetin-supplemented groups by 8–18%, as compared with the control group.
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Table 3 Effect of naringenin supplementation on egg production and quality in laying hens. Variables
Naringenin (g/kg)
Egg production (%) Eggshell strength (g/cm2 ) Eggshell thickness (mm) Egg weight (g) Yolk weight (g) Yolk weight/egg weight Cholesterol/g yolk (mg/g) Total cholesterol/egg (mg/g)
SEM
0
0.5
1
2
4
94.6bc 3.11 0.45 56 16.3b 0.29 11.8a 192a
95.9ab 3.13 0.46 59 16.2b 0.30 10.7b 173b
96.3a 3.14 0.47 55 16.5ab 0.30 10.9b 181b
95.8ab 3.14 0.47 58 17.4a 0.30 9.1c 158c
93.3c 3.13 0.46 56 16.5ab 0.29 10.8b 178b
1.1 0.1 0.02 1.1 0.5 0.1 0.1 2.4
P value Linear
Quadratic
0.14 0.11 0.29 0.68 0.15 0.16 0.11 0.12
<0.01 0.21 0.21 0.29 <0.01 0.15 <0.01 <0.01
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). Table 4 Effect of hesperetin and naringenin supplementation on the serum traits in laying hens. Variables
0 g/kg
0.5 g/kg
1 g/kg
2 g/kg
4 g/kg
SEM
P value Linear
Quadratic
Hesperetin Cholesterol (mg/dl) Triglyceride (mg/dl)
176ab 1647ab
151ab 1538ab
137bc 1496ab
110c 1160b
116c 1236b
25.3 207.6
0.07 0.44
0.04 0.03
Naringenin Cholesterol (mg/dl) Triglyceride (mg/dl)
169a 1990a
111c 1529ab
150ab 1629ab
121bc 1133b
141abc 1603ab
23.1 305.4
0.22 0.25
<0.01 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05).
Furthermore, results indicated that naringenin supplementation at 2 g/kg was sufficient to exert a significant hypocholesterolemic effect on egg yolk in laying hens (P<0.05) (Table 3). The cholesterol/g yolk and total cholesterol/egg in naringenin-supplemented group was also reduced (P<0.05) at 12–22%, and showed a quadratic dose response. 3.4. Effect of hesperetin and naringenin on serum traits Results from both experiments showed that the supplementation of hesperetin at 2 and 4 g/kg, and naringenin at 0.5 and 2 g/kg were significantly lowered (P<0.05) the serum total cholesterol concentration than the control group. The supplementation of hesperetin at 2 and 4 g/kg, and naringenin at 2 g/kg were significantly lowered (P<0.05) the serum triglyceride concentration as compared to the control group (Table 4). These groups also displayed a quadratic dose response. 3.5. Effect of hesperetin and naringenin on antioxidant activities Comparing the antioxidant enzyme activities with the control group, SOD, catalase, scavenging O2 − and iron-chelating ability were higher (P<0.05) in the 2 g/kg hesperetin-supplemented group, while the 2 g/kg naringenin-supplemented group increased (P<0.05) SOD, catalase, trolox equivalent antioxidant capacity, scavenging O2 − and iron-chelating ability. Hesperetin and naringenin supplementation at 0.5 g/kg level was significant differences in SOD activity, and at 2 g/kg level was the highest. Most of them showed quadratic dose responses, except for iron-chelating in hesperetin and trolox equivalent antioxidant capacity in naringenin, which showed a linear dose response. There were no differences in the GSH Px at all levels in both naringenin- and hesperetin-supplemented groups (Tables 5 and 6). 4. Discussion 4.1. Effects of hesperetin and naringenin on egg quality and production The results of this study showed the potential of hesperetin and naringenin to lower egg yolk cholesterol content. Lien et al. (2008) also reported reduced egg yolk cholesterol content when adding 0.5 g/kg of hesperetin and naringenin to the layers’ diet. Egg yolk cholesterol may be reduced due to the inhibition of HMG-CoA reductase activity, the key enzyme that regulates the rate of cholesterol biosynthesis in the liver, thus inhibiting the enzyme can reduce cholesterol synthesis (Lee et al., 1999, 2003; Bok et al., 2000; Kim et al., 2003). The cholesterol content of the yolk is primarily dependent on the cholesterol content of vitellogenin and triglyceride-rich lipoproteins-VLDL. Virtually all of the cholesterol found in the egg yolk is originated in the liver, where it is synthesized, and transported in the plasma to the ovaries where they are taken up into the developing follicles by receptor-mediated endocytosis.
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Table 5 Effect of hesperetin supplementation on anti-oxidant activity in laying hens. Variables
SOD (n mol/mg protein) Catalase (n mol/mg protein) GSH Px (n mol/mg protein) Trolox equivalent antioxidant capacity (mM/l) Iron-chelating (%) Scavenging O2 − (%)
Hesperetin (g/kg)
SEM
0
0.5
1
2
4
23.5c 73.3b 20.0 38.1 76.1b 84.4b
41.5ab 81.8a b 21.6 38.7 80.1ab 86.6ab
42.6a 86.7a b 20.9 37.6 87.1a 88.3ab
43.7a 88.6a 21.8 36.3 86.8a 94.3a
34.5ab 86.5a b 21.1 33.7 87.1a 86.5ab
8.4 6.2 0.7 2.1 5.1 3.7
P value Linear
Quadratic
0.28 0.09 0.08 0.08 0.02 0.11
0.04 0.02 0.16 0.12 0.10 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). SOD: superoxide dismutase. GSH Px: glutathione peroxidase. Table 6 Effect of naringenin supplementation on anti-oxidant activity in laying hens. Variables
SOD (n mol/mg protein) Catalase (n mol/mg protein) GSH Px (n mol/mg protein) Trolox equivalent antioxidant capacity (mM/l) Iron-chelating (%) Scavenging O2 - (%)
Naringenin (g/kg)
SEM
0
0.5
1
2
4
24.7c 78.3b 20.3 34.2b 77.9b 84.5b
37.7b 83.1ab 21.8 45.9ab 83.4ab 87.5ab
43.2ab 89.4ab 22.0 47.5ab 81.9ab 87.2ab
46.6a 91.9a 22.6 53.9a 87.7a 94.2a
40.1ab 83.0ab 22.5 54.5a 86.0ab 86.4ab
2.6 4.7 0.9 8.6 3.8 2.8
P value Linear
Quadratic
0.12 0.08 0.11 0.04 0.07 0.08
0.02 0.01 0.19 0.16 <0.01 0.03
n = 20. a, b, c Means in the same row with different superscripts are significantly different (P<0.05). SOD: superoxide dismutase. GSH Px: glutathione peroxidase.
The maximum reduction of egg yolk cholesterol by hesperetin and naringenin was 18% and 22%, respectively, although the percentage reduction was less than those reported for triparanol (77%), azasterols (80%) and atorvastatin (46%) (Dam et al., 1979; Cecil et al., 1981; Elkin et al., 1999). However, triparanol and azasterols had serious side effects; and atorvastatin largely reduced egg production (Elkin, 2006, 2007). Hesperetin and naringenin are natural organic flavonoids compounds and displayed antioxidant capacity in this study, and in other reports (Jeon et al., 2001; Seo et al., 2003; Kim et al., 2004). Some investigations compared hesperetin and naringenin with lovastatin – a cholesterol reducing medicine, and found that they had comparable effects (Kim et al., 2003). Thus, the use of hesperetin and naringenin to produce low cholesterol egg production may more acceptable to consumers. Furthermore, the extractions of hesperitin and naringenin from orange and grapefruit peels are simple and inexpensive. There were no effects of hesperetin and naringenin on eggshell strength and eggshell thickness. The level of lime (26 g/kg) present in the hesperetin and naringenin could have influenced these measurements as lime has been shown previously to influence eggshell quality (Plimmer and Lowndes, 1924). However this did not occur in our study, and furthermore, there are no reports showing that lime content in the diet influences the serum and yolk cholesterol contents or antioxidant capacity. 4.2. Effects of hesperetin and naringenin on serum traits This study has demonstrated that both hesperetin and naringenin supplementation (2 g/kg) could significantly lower the serum cholesterol concentration, corresponding to other findings (Lee et al., 2003; Seo et al., 2003; Kim et al., 2004; Lien et al., 2008). Similarly, serum triglyceride was significantly lowered in 2 g/kg hesperitin and naringenin supplementation. This finding is consistent with Lee et al. (1999, 2003), Bok et al. (2000), and Lien et al. (2008). The reduction of serum cholesterol could be due to the inhibition of the key enzymes in the cholesterol synthesis, HMG-CoA reductase. The blockage of cholesterol synthesis results in a lower intracellular supply of cholesterol. Wilcox et al. (1998) and Borradaile et al. (1999) reported that hesperetin and naringenin not only decreased cholesterol synthesis but also inhibited ACAT activity in HepG2 cells. Inhibition of ACAT reduced the hepatic production of apo-B containing lipoproteins (Burnett et al., 1998) such as VLDL, thereby reducing the plasma triglyceride concentration. 4.3. Effects on antioxidant activities This study showed that SOD level was relatively high for hesperetin and naringenin at all supplementation levels; even a low level (0.5 g/kg) of supplementation was able to exert high serum SOD activity. The results correspond with a similar study on the supplementation of hesperetin and naringenin by Lien et al. (2008) on layer hens. Furthermore, Jeon et al. (2001), Seo et al. (2003) and Kim et al. (2004) also reported a high level of hepatic SOD activity in naringenin-fed rabbits,
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rats and mice. Catalase activity was increased at 2 g/kg supplementation level for both hesperetin and naringenin. Jeon et al. (2001) and Kim et al. (2004) also found that catalase activity was increased in rabbits and mice fed with 0.5 g/kg or 0.2 g/kg naringenin. The increased SOD activity by hesperetin and naringenin supplementation resulted in a reduced superoxide anion level. GSH-Px activity remained low, probably due to higher SOD activity that might have led to a reduced peroxide level in the serum. Trolox equivalent antioxidant capacity showed no difference in all hesperetin supplementation levels while higher activity was detected at 2 and 4 g/kg naringenin supplementation. Scavenging O2 − activity was higher at both 2 g/kg of hesperetin- and naringenin-supplemented groups, which is consistent with the study of Chen et al. (1990). Lien et al. (2008) also found that hesperetin and naringenin have a strong superoxide free radical scavenging activity. The results suggested that hesperetin and naringenin can terminate chain radical reaction by donating hydrogen atoms to the peroxy hydroxyl (Cook and Samman, 1996). Since both hesperetin and naringenin are rich in OH groups to supply hydrogen atoms for free radicals to block the oxidation chain reaction, both hesperetin and naringenin have antioxidant activity. Egg production was reduced in the 4 g/kg hesperetin-supplemented group, indicating that the supplementation level of 4 g/kg is too high; but the reason remains unknown. According to the results on yolk cholesterol, serum traits and antioxidant activity, the supplemental levels in most results displayed a quadratic dose response and indicated that the supplementation level of 2 g/kg for both hesperetin and naringenin seemed to be the optimum level for reducing serum and yolk cholesterols contents and enhancing antioxidant capacity. 5. Conclusion Data from both experiments suggests that both hesperetin and naringenin can lower plasma and egg yolk cholesterol levels and improve the antioxidant capacity. Our data suggests that the optimum level of supplementation for both hesperetin and naringenin is 2 g/kg in the basal diet for a reduction in serum and yolk cholesterols contents and enhanced antioxidant capacity, without impairment of egg production. References Bhat, G.B., Tinsley, S.B., Tolson, J.K., Patel, J.M., 1992. Hypoxia increases the susceptibility of pulmonary artery endothelial cells to hydrogen peroxide injury. J. Cell. Physiol. 151, 228–238. Bok, S.H., Shin, Y.W., Bae, K.H., Jeong, T.S., Kwon, Y.K., Park, Y.B., Choi, M.S., 2000. Effects of Naringenin and Lovastatin on plasma and hepatic lipids in high fat and high-cholesterol fed rat. Nutr. Res. 20, 1007–1015. Borradaile, N.M., Carroll, K.K., Kurowska, E.M., 1999. 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