The Effect of Essential Oil Intake on Changes of Plasma Antioxidant Status in Mice

Faix Š., Š. Juhas, Z. Faixová: The Effect of Essential Oil Intake on Changes of Plasma Antioxidant Status in Mice. Acta Vet. Brno 2007, 76: 357-361. The aim of this study was to determine the effects of four essential oils intake by feed, namely Origanum vulgare, Thymus vulgaris, Cinnamomum zeylanicum Ness, and Syzygium aromaticum on antioxidant status in mice in vivo. Essential oils were in the aether oleum form. They were diluted with ethanol absolute mixed with ground pelett (0.1, 0.25, 0.57 and 1% concentration) and thereafter ethanol was evaporated. SOD, GPx activities and TAS were measured in erythrocytes and plasma spectrophotometrically with Ransod, Ransel and TAS kits from RANDOX, respectively. GPX activity showed a signifi cant increase in 0.25% and 0.1% concentration of Origani aetheroleum. The GPx activities were decreased in 1% concentration of Thymi aetheroleum and 0.57% concentration of Cinnamomi aetheroleum and 0.57% concentration of Caryophylli aetheroleum. The total antioxidant status showed a signifi cant decrease in 1 % concentration of Origani aetheroleum and signifi cantly increased in 0.1% concentration. The same results were found in Thymi aetheroleum. Cinnamomi aetheroleum and Caryophylli aetheroleum had not effect on total antioxidant status. SOD activities were not signifi cantly changed after intake of essential oils. In conclusion, our results showed, that concentration of essential oil is very important for antioxidant status and also for metabolism of mice, because a high dose of essential oil has adverse effect on metabolism of mice, representated by a lower growth of the body weight. On the other hand, essential oils at lower concentrations have positive effect on antioxidant status of mice. Antioxidant activity, vegetable essence, GPx, SOD, TAS Antioxidant systems are being shown to play an increasing role in the protection against exogenous oxidative stress. Many authors have reported antimicrobial, antifungal, antioxidant and radical-scavenging properties by spices and essential oils and, in some cases, a direct food-related application has been tested (Madsen and Bertelsen 1995; Sawamura 2000; Horosova et al. 2004; Hsu and Liu 2004a; Masella et al. 2004; Sacchetti et al. 2005). Spices and herbs are recognized as sources of natural antioxidants and thus play an important role in the chemoprevention of diseases. Exposure to oxidant molecules released from the environment, nutrition or pathologies can generate reactive oxygen species (Faix et al. 2005). Cells have developed enzymatic systems that convert oxidants into non-toxic molecules, thus protecting the organism from the deleterious effects of oxidative stress. SOD is the fi rst line in cell defense against oxidative stress. It converts the superoxide anion O2 into a less toxic product, namely H2O2 and O2 (McCrod et al. 1976). In H2O2 detoxifi cation, the selenium dependent glutathione peroxidase (GSHPx) converts H2O2 into water via the oxidation of reduced glutathione (GSH) in oxidized glutathione (GSSG) (Bruce et al. 1982). The extracts from Origanum vulgare, Thymus vulgaris, Cinnamomum zeylanicum Ness and Syzygium aromaticum were evaluated for the possible mode of action by studying their antioxidant potential. Materials and Methods During the whole study, the principles of the Ethical committee for the protection of animals in research of the Institute were strictly followed. ACTA VET. BRNO 2007, 76: 357-361; doi:10.2754/avb200776030357 Address for correspondence: Doc. MVDr. Štefan Faix PhD. Institute of Animal Physiology Slovak Academy of Sciences Šoltesovej 4-6 040 01 Košice, Slovak Republic Phone:++421 55 63 362 68 Fax: ++421 55 63 320 48 e-mail: faix@saske.sk http://www.vfu.cz/acta-vet/actavet.htm Four essential oils, namely Origanum vulgare, Thymus vulgaris, Cinnamomum zeylanicum Ness and Syzygium aromaticum were obtained from CALENDULA a.s. (Nová Ľubovňa, Slovak Republic). Ransod, Ransel and TAS kits were obtained from RANDOX Laboratories Ltd, UK. Male mice (ICR bred), 5-6 weeks of age, were used in the present study. The mice were housed at 22 °C with a 12:12-h dark–light cycle (5.00–17.00 hours lights on). They were maintained on a standard pellet diet for mice with tap water ad libitum. Each group contained 10 mice. The control groups were given standard diet for 20 days. Experimental groups were fed standard diet plus supplementation of different essential oils. Essential oils were in aether oleum form. They were diluted with ethanol absolute mixed with ground pellet and thereafter ethanol was evaporated. Final content concentration was different for each essential oil. The fi nal contents of Origani aetheroleum were 1%, 0.57%, 0.25% and 0.1%. The fi nal contents of Thymi aetheroleum were 1%, 0.25% and 0.1%. Cinnamomi aetheroleum and Caryophylli aetheroleum were used at 0.57% concentration only. Mice were removed from their cage and rapidly decapitated by guillotine. Blood samples were collected into tubes containing heparin. For SOD determinations, erythrocytes were obtained from 1 ml of blood by centrifugation at 805 g for 10 minutes, at room temperature, immediately after the blood was drawn; they were washed three times in a 0.9 mol NaCl solution and stored at -70 °C until analysis. For GPx activity assays, fresh whole blood was collected and stored at -70 °C. Samples were haemolysed by the addition of ice cold distilled water (1/10), cell membranes were removed by centrifugation, and the supernatant was used for the analysis. SOD (EC 1.15.1.1) and GPx (EC 1.11.1.9) activities were assayed in erythrocytes spectrophotometrically with Ransod and Ransel kits, respectively, using a UVVIS spectrophotometer. SOD activity was expressed as the amount of protein causing a 50% inhibition of formazan dye (505 nm), employing xanthine and xanthine oxidase to generate superoxide radicals. Units of GPx activity were calculated following NADPH oxidation at 340 nm using cumene hydroperoxide as the substrate. Total antioxidant status was measured in blood plasma by incubation of ABTS with a peroxidase (metmyoglobin) and hydrogen peroxide in the production of the radical cation ABTS• +. This species is blue-green in colour and can be detected at 600 nm. Data are expressed as mean ± (S.E.M.). The comparison between values was performed by unpaired Studentُs t-test. Results and Discussion The present study evaluated the effects of feeding diets with essential oils contents on oxidative stress in mice. As shown in Fig. 1, GPx activity showed a signifi cant increase in 0.25% and 0.1% concentration of Origani aetheroleum (388.7 ± 2.7 and 366.1 ± 4.6 vs 329.5 ± 1.6 U·g-1 Hb, P < 0.001). The GPX activities were decreased in 1% concentration of Thymi aetheroleum (457.7 ± 8.7 vs 505.2 ± 11.8 U·g-1 Hb, P < 0.05) and 0.57% concentration of Cinnamomi aetheroleum (352.0 ± 16.5 vs 434.8 ± 13.3 U·g-1 Hb, P < 0.01) and 0.57% concentration of Caryophylli 358 Fig. 1. GPx concentration in blood of mice. Each value represents the mean ± S.E.M.; n = 10 (*P < 0.05, **P < 0.01, ***P < 0.001) aetheroleum (318.1 ± 34.48 vs 434.8 ± 13.28 U·g-1 Hb, P < 0.01). The reducing activities of GPx at high concentration of essential oils may be a result of pro-oxidative potential. Hodgson and Fridovich (1975) indicated that reduced activities of GPx may result from radical-induced inactivation and glycation of the enzyme. SOD activities were not signifi cantly changed after essential oils intake. It is possible that the antioxidant properties of essential oils are being utilised by the cells, thus sparing the intracellular antioxidant system. It is also possible that essential oils infl uenced other cellular systems suggesting that more detailed examination of antioxidant parameters is required. The protective role of essential oils may result from its antioxidative defense mechanism through the induction of antioxidant enzyme activities. A single dose of sesame oil may attenuate oxidative stress (Hsu and Liu 2004b). As Fig. 2 shows, total antioxidant status showed a signifi cant decrease in 1 % concentration of Origani aetheroleum (0.220 ± 0.02 vs 0.315 ± 0.026 mmol·l-1, P < 0.05) and signifi cant increase in 0.1% concentration (0.389 ± 0.042 vs 0.241 ± 0.026 mmol·l-1, P < 0.05). The total antioxidant status was decreased in 1% concentration of Thymi aetheroleum (0.036 ± 0.009 vs 0.158 ± 0.015 mmol·l-1, P < 0.001) and increased in 0.1% concentration (0.286 ± 0.04 vs 0.158 ± 0.015 μmol·l-1, P < 0.05). Cinnamomi aetheroleum and Caryophylli aetheroleum had no effect on total antioxidant status. Our fi ndings show evidence of an increased sensitivity to oxidative stress at high dose intake of some vegetable extracts. The intake of medicinal plants in rats results in an increase in antioxidant enzyme activity and a decrease in malondialdehyde, which may reduce the risk of infl ammation (Choi and Hwang 2005). Hypericum perforatum and Calendula offi cinalis hydroalcoholic extracts showed a signifi cant activity (Herold et al. 2003). It will be necessary to fi nd molecules that will increase directly or indirectly the level of antioxidant system. Our results show that essential oils affected oxidative stress in organism and they have positive and negative effect on antioxidant status. In diabetic rats treated with the ethanolic extract, a signifi cant increase in activity of antioxidant enzymes was observed. This might refl ect the antioxidant potency of the ethanolic extract, which by reducing blood glucose levels prevented 359 Fig. 2. Total antioxidant status of blood in mice. Each value represents the mean ± S.E.M; n = 10 (*P < 0.05, ***P < 0.001)

Antioxidant systems are being shown to play an increasing role in the protection against exogenous oxidative stress.Many authors have reported antimicrobial, antifungal, antioxidant and radical-scavenging properties by spices and essential oils and, in some cases, a direct food-related application has been tested (Madsen and Bertelsen 1995;Sawamura 2000;Horosova et al. 2004;Hsu and Liu 2004a;Masella et al. 2004;Sacchetti et al. 2005).Spices and herbs are recognized as sources of natural antioxidants and thus play an important role in the chemoprevention of diseases.Exposure to oxidant molecules released from the environment, nutrition or pathologies can generate reactive oxygen species (Faix et al. 2005).Cells have developed enzymatic systems that convert oxidants into non-toxic molecules, thus protecting the organism from the deleterious effects of oxidative stress.SOD is the fi rst line in cell defense against oxidative stress.It converts the superoxide anion O 2 -into a less toxic product, namely H 2 O 2 and O 2 (McCrod et al. 1976).In H 2 O 2 detoxifi cation, the selenium dependent glutathione peroxidase (GSHPx) converts H 2 O 2 into water via the oxidation of reduced glutathione (GSH) in oxidized glutathione (GSSG) (Bruce et al. 1982).
The extracts from Origanum vulgare, Thymus vulgaris, Cinnamomum zeylanicum Ness and Syzygium aromaticum were evaluated for the possible mode of action by studying their antioxidant potential.
Male mice (ICR bred), 5-6 weeks of age, were used in the present study.The mice were housed at 22 °C with a 12:12-h dark-light cycle (5.00-17.00hours lights on).They were maintained on a standard pellet diet for mice with tap water ad libitum.Each group contained 10 mice.The control groups were given standard diet for 20 days.Experimental groups were fed standard diet plus supplementation of different essential oils.Essential oils were in aether oleum form.They were diluted with ethanol absolute mixed with ground pellet and thereafter ethanol was evaporated.Final content concentration was different for each essential oil.The fi nal contents of Origani aetheroleum were 1%, 0.57%, 0.25% and 0.1%.The fi nal contents of Thymi aetheroleum were 1%, 0.25% and 0.1%.Cinnamomi aetheroleum and Caryophylli aetheroleum were used at 0.57% concentration only.Mice were removed from their cage and rapidly decapitated by guillotine.Blood samples were collected into tubes containing heparin.For SOD determinations, erythrocytes were obtained from 1 ml of blood by centrifugation at 805 g for 10 minutes, at room temperature, immediately after the blood was drawn; they were washed three times in a 0.9 mol NaCl solution and stored at -70 °C until analysis.For GPx activity assays, fresh whole blood was collected and stored at -70 °C.Samples were haemolysed by the addition of ice cold distilled water (1/10), cell membranes were removed by centrifugation, and the supernatant was used for the analysis.
SOD (EC 1.15.1.1)and GPx (EC 1.11.1.9)activities were assayed in erythrocytes spectrophotometrically with Ransod and Ransel kits, respectively, using a UV-VIS spectrophotometer.SOD activity was expressed as the amount of protein causing a 50% inhibition of formazan dye (505 nm), employing xanthine and xanthine oxidase to generate superoxide radicals.Units of GPx activity were calculated following NADPH oxidation at 340 nm using cumene hydroperoxide as the substrate.Total antioxidant status was measured in blood plasma by incubation of ABTS with a peroxidase (metmyoglobin) and hydrogen peroxide in the production of the radical cation ABTS• +.This species is blue-green in colour and can be detected at 600 nm.
Data are expressed as mean ± (S.E.M.).The comparison between values was performed by unpaired Student ُ s t-test.
SOD activities were not signifi cantly changed after essential oils intake.It is possible that the antioxidant properties of essential oils are being utilised by the cells, thus sparing the intracellular antioxidant system.It is also possible that essential oils infl uenced other cellular systems suggesting that more detailed examination of antioxidant parameters is required.
The protective role of essential oils may result from its antioxidative defense mechanism through the induction of antioxidant enzyme activities.A single dose of sesame oil may attenuate oxidative stress (Hsu and Liu 2004b).
As Fig. 2 shows, total antioxidant status showed a signifi cant decrease in 1 % concentration of Origani aetheroleum (0.220 ± 0.02 vs 0.315 ± 0.026 mmol•l -1 , P < 0.05) and signifi cant increase in 0.1% concentration (0.389 ± 0.042 vs 0.241 ± 0.026 mmol•l -1 , P < 0.05).The total antioxidant status was decreased in 1% concentration of Thymi aetheroleum (0.036 ± 0.009 vs 0.158 ± 0.015 mmol•l -1 , P < 0.001) and increased in 0.1% concentration (0.286 ± 0.04 vs 0.158 ± 0.015 μmol•l -1 , P < 0.05).Cinnamomi aetheroleum and Caryophylli aetheroleum had no effect on total antioxidant status.Our fi ndings show evidence of an increased sensitivity to oxidative stress at high dose intake of some vegetable extracts.The intake of medicinal plants in rats results in an increase in antioxidant enzyme activity and a decrease in malondialdehyde, which may reduce the risk of infl ammation (Choi and Hwang 2005).Hypericum perforatum and Calendula offi cinalis hydroalcoholic extracts showed a signifi cant activity (Herold et al. 2003).It will be necessary to fi nd molecules that will increase directly or indirectly the level of antioxidant system.Our results show that essential oils affected oxidative stress in organism and they have positive and negative effect on antioxidant status.In diabetic rats treated with the ethanolic extract, a signifi cant increase in activity of antioxidant enzymes was observed.This might refl ect the antioxidant potency of the ethanolic extract, which by reducing blood glucose levels prevented glycation and inactivation of enzymes (Rajasekaran et al. 2005).Similar kinds of effect, i.e. prevention of potential glycation of antioxidant enzymes and the ensuing decrease in activity, have been reported with other plants, such as Eugenia jambolana, well-known for their antidiabetic activity (Ravi et al. 2004).
As Table 1 shows, body weights were signifi cantly lower in 14 day of 1% Origani aetheroleum intake (23.41 ± 0.5 vs 27.85 ± 0.4 g, P < 0.001) and 7 day of 1% Thymi aetheroleum intake (17.4 ± 0.5 vs 21.08 ± 0.4 g, P < 0.05).A similar rise in body weight was observed in control and experimental groups of mice with age.Youdim and Deans (1999) show, that increase in body weight of control rats did not differ signifi cantly from that of thyme oil treated rats except at 22 months of age, where control body weights were found to be signifi cantly higher.
A number of previous reports suggest that cellular antioxidants are under homeostatic control and that dietary antioxidant supplementation depresses endogenous antioxidant synthesis so as to nullify the expected benefi cial effect of the supplement (Barja de Quiroga et al. 1992;Bunker 1992;Warner 1992).
The oral administration of low doses to mice reduces oxidative stress induced by higher levels of antioxidant enzymes in the plasma.Thus, it appears that these spices exert antioxidant protection through their ability to activate the antioxidant enzymes.