The purpose of present study was to investigate the effect of rosemary and garlic oils on the lipid peroxidation and glutathione redox system in the blood and liver of broiler chicken. Day-old Hubbard broiler chickens (n=200) were fed with commercial broiler feed (control) and supplemented with garlic oil (0.25 g kg
-1), rosemary oil (1.5 g kg
-1) or their combination (0.25 g kg
-1 garlic oil and 1.5 g kg
-1 rosemary oil). At the end of the growing period (42 days of age) blood and liver samples of 10 animals were taken from each group to determine malondialdehyde and reduced glutathione content and glutathione peroxidase activity. There were no significant differences in the blood plasma or in red blood cell haemolysates among the groups, but garlic oil supplementation increased significantly reduced glutathione content and both essential oils the glutathione peroxidase activity in liver. However, combination of the two oils caused increase of malondialdehyde content of liver together with significantly higher glutathione peroxidase activity as compared to the control. Due to the beneficial effect on glutathione redox system both essential oils - used solely - can be used to reduce the effects of oxidative processes in physiological conditions
Acta Biol Szeged 53(Suppl.1): (2009)
Phytobiotics or essential oils have importance as natural antioxidants both in animal and human nutrition but their antioxidant capacity is measurable in comparison with synthetic antioxidants only at relatively high doses (Cuvelier et al. 1990) and it was proven mostly only in Fe
2+/ascorbate system (Bozin et al. 2006, 2007). The main biologically active components of rosemary oil are carnosol, carnosic acid and its esters (Boutekedjiret et al. 2003). Some previous experiments showed that phenolic compounds of rosemary oil, such as carnosic acid act as antioxidants such as
α-tocopherol (McCarthy et al. 2001) and its antioxidant activity was higher than some synthetic antioxidants in vitro (Richheimer et al. 1996). It was also found that rosemary oil improve meat (McCarthy 2001; Smet et al. 2005; Govaris et al. 2007) and egg (Galobart et al. 2001) quality, improve the resistance of the polyunsaturated fatty acids and cholesterol against oxidative damage.
The main active component of garlic oil is alliin (S-aly-lylcysteine sulfoxide) or its derivatives which are showed antioxidant capacity in vitro (Yamasaki et al. 1994). Among the different, lipid soluble organic sulphur components of garlic, such as diallyl-disulphide (DADS), S-ethyl-cysteine or n-acetyl-cysteine (NAC) also have antioxidant properties in vitro (Dwivedi et al. 1998) and significantly decreased the rate of lipid oxidation and oxymioglobin formation in minced beef which also proved their antioxidant properties (Yin and Cheng 2003).
Materials and Methods
A total of 200 day-old Hubbard broiler chickens were divided randomly into four treatment groups. Control group was fed with commercial diet without added antioxidants, while the feed of the treatment groups supplemented with 1.5 g kg
-1 rosemary oil, 0.25 g kg
-1 garlic oil or combination of them (1.5 g kg
-1 rosemary oil and 0.25 g kg
-1 garlic oil), respectively. The feeding trial lasted for 42 days and at the end of growing period 10 animals from each group was exterminated and blood and liver samples were taken.
Blood samples were taken after cervical dislocation into EDTA-Na
2 containing tubes. Blood plasma and blood cells were separated by centrifugation (2500 g, 20 min). Red blood cells were haemolysed with 1:9 volume of redistilled water and by freezing (-20ºC, 18 hours) and thawing (25ºC, 30 min). Liver samples were taken from the distal part of the right lobe and store at -18ºC until analysis. Liver homogenates were made with 9-fold cold (4ºC) physiological saline (0.65 % w/v NaCl). Determination of malondialdehyde content was carried out from the crude homogenate, while other parameters determined from the 10,000 g supernatant fraction of the homogenate.
Malondialdehyde content of blood plasma and red blood cells (RBC) haemolysate was determined according to Placer et al. (1966) as modified by Matkovics et al. (1988) while in liver homogenate according to Mihara et al. (1980). Reduced glutathione (GSH) content of blood plasma, RBC haemolysate and liver homogenate was measured as described by Ancsin et al.
Table 1. Malondialdehyde and reduced glutathione content and glutathione peroxidase activity of blood plasma of chickens fed with different essential oils (mean
±S.D.).
Table 2. Malondialdehyde and reduced glutathione content and glutathione peroxidase activity of RBC haemolysate of chickens fed with different essential oils (mean
±S.D.).
Sedlak and Lindsay (1968), while glutathione peroxidase (E.C. 1.11.1.9) according to Matkovics et al. (1988). Enzyme activity expressed to protein content which was determined by biuret method (Weichselbaum 1948) for blood plasma and RBC haemolysate or with Folin-phenol reagent for liver homogenate (Lowry et al. 1951).
Statistical evaluation of the data was performed using paired LSD test (Statistica“ 4.5, Statsoft Inc., 1993).
Results and Discussion
There were no significant differences in any of the measured parameters in blood plasma or RBC haemolysate (Tables 1 and 2). These results can be explained with the fast absorption, metabolic conversion and excretion of essential oil constituents that was mentioned by Kohlert et al. (2000). Additionally these results suggest that the effect of essential oils manifested mainly in tissues, but not in blood, possibly because of their lipid soluble characteristics. Lee et al. (2004) reported that in spite of the short half-life of the essential oils, those are able to accumulate in chicken tissues - mainly in liver and kidney - if the administration is continuous without withdrawal periods.
Malondialdehyde content of liver homogenates were not modified by the two essential oils if those were applied separately. However, combined treatment caused significantly higher malondialdehyde concentration (Table 3). That result can be explained by the findings of Skibola and Smith (2000) and also with those of Liu (2003), who found that excess amount of essential oils in the diet, may have not have antioxidant but pro-oxidant effect.
Glutathione content was significantly higher in liver homogenate as effect of garlic oil supplementation (Table 3). There are two explanation of that result. First, garlic oil contains organic sulphur compounds which may react with 5,5'-dithiobis-2 nitrobensoic acid used as colour complex reagent for the determination of reduced glutathione as non-protein sulphydryl group. Second, garlic oil prevents oxidation of glutathione through its active components, such as S-allylcysteine, that lead to functional recovery (Sener et al. 2007). Additionally garlic oil possibly improves the absorption of amino acids, such as methionine or cysteine, from the gastro-intestinal tract which are the limiting factors of glutathione synthesis (Wang et al. 1997).
Glutathione peroxidase activity was significantly higher in the 10,000 g supernatant fraction of liver homogenates of all three treated groups as compared to the control (Table 3). Glutathione peroxidase activity depends on the presence of its active centre, selenium (Sun et al. 1998), also its substrates, reduced glutathione (Németh et al. 2004) and oxygen free radicals which are first increase and at continuously high level decrease the enzyme activity (Holovska et al. 1996). The results suggest that garlic or rosemary oil as antioxidants decrease the oxygen free radical formation therefore increase the enzyme activity and the same effect was found if those were applied together, and based on the significantly higher malondialdehyde content, provoke free radical formation but only at low extent which increase the enzyme activity. Auroma et al. (1992) reported that carnosic acid appears to scavenge H
2O
2, but it could also act as a substrate for the peroxidase system, also there are some reports about the possible pro-oxidant effects of some garlic oil constituents Amagase et al. 2001).
Table 3. Malondialdehyde and reduced glutathione content and glutathione peroxidase activity of liver homogenates of chickens fed with different essential oils (mean
± S.D.).
In conclusion, the results showed that both garlic and rosemary oils have antioxidant properties and those have positive effect on the glutathione redox system of liver in chickens, but using together in a much higher amount those have opposite, pro-oxidant, effect.
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