EFFECTS OF haBeRlea RhoDoPensis EXTRACT ON ANTIOXIDATION AND LIPID PEROXIDATION IN RABBITS AFTER EXPOSURE TO 60CO-γ-RAYS

Haberlea rhodopensis extract (HRE) possesses strong antioxidant activity. The aim of the present study was to evaluate the protective ability of HRE against oxidative damage induced by a non-lethal dose of 60Co-γ-rays. Experimental animals (New Zealand rabbits) were exposed to 2.0 Gy γ-rays before and after HRE administration. Lipid peroxidation (MDA) and activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were analyzed. Results show that administration of HRE before and after irradiation decreased the MDA level and increased SOD and CAT activity, thus providing protection against the radiation-induced decrease in antioxidative ability and increase in lipid peroxidation. This finding supports the idea that HRE is a potent free radical scavenger and antioxidant.


INTRODUCTION
Reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anion radicals (O 2 -), and hydrogen peroxide (H 2 O 2 ), produced during normal metabolism or as a consequence of response to abnormal stress, are implicated in the pathogenesis of aging and diseases, including cancer (Ho et al., 1998).Mammalian cells are equipped with both enzymatic and non-enzymatic antioxidant mechanisms to minimize cellular damage resulting from the interaction between cellular constituents and ROS (Forman and Fisher, 1982;Spitz et al., 2004).The enzymatic antioxidant mechanism involves enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) as well as the enzymes involved in the recycling of oxidized glutathione (GSH) such as glutathione reductase.Although endogenous cellular antioxi-dants act in concert to eliminate ROS accumulation in a physiological state under pathological conditions, ROS overload might exceed the cellular antioxidant capacity, affecting critical biological macromolecules and triggering oxidative stress (Halliwell, 1992).Exposure to ionizing radiation produces significant alterations in oxidant activity in different tissues (Hui et al., 1996).The endogenous antioxidant defenses are inadequate to cope with induced damage.Appropriate antioxidant intervention seems to inhibit or reduce free radical toxicity and thus offers protection against radiation (Greenstock, 1993).
The study of medicinal plants in traditional medicine as modifiers of radiation effects is a new area of research.Many such medicinal plants have hepatoprotective, neuroprotective, anti-inflammatory and also antioxidant or radical-scavenging properties (In et al., 2000;Park et al., 1993;Suja et al., 2004).Haberlea rhodopensis Friv.(Gesneriaceae family) is a Balkan endemite belonging to the group of extremely desiccation-tolerant (resurrection) plants which are capable of withstanding long periods of almost full desiccation and to recover quickly on water availability (Markovska et al., 1994;Georgieva et al., 2008).Carbohydrates and phenols were found to play an important role in the survival of plants under extreme conditions (Muller et al., 1997).Phenolic compounds, accumulated in high amounts in resurrection plants, are assumed to protect their membranes against desiccation and free radical-induced oxidation (Sgherri et al., 2004).
Ethnobotanical data showed that Haberlea leaves were used for the treatment of wounds and diseases of stock in the Rhodope region of Bulgaria.One of the local plant names in the Rhodope Mountains is "shap" (food and mouth disease), which is considered as confirmation that the local people were using the plant against animal diseases.
The present study was conducted to elucidate the radioprotective potential of total extract of the "resurrection" plant Haberlea rhodopensis in rabbits using the activity of antioxidant enzymes SOD, CAT and the level of MDA as an experimental endpoint.

Leaf extract of Haberlea rhodopensis
Fresh leaves of Haberlea rhodopensis (Friv.)were collected from their natural habitat (in the vicinity of Asenovgrad, Bulgaria) during flowering in May-June.They were botanically identified in the Department of Pharmacology and Pharmacognosy, (Medical University, Sofia, Bulgaria) by a botanistphytotherapist.
Leaves were cut into small pieces and dried at room temperature for 1 month.After grinding, the dry matter was macerated for 6 h in 70% ethyl alcohol and percolated for 48 h.Primary extract was concentrated by evaporation of ethanol in a vacuum environment to reach a ratio of 5% ethanol and 95% water.The obtained extract was filtered through filter paper to remove emulsified substances, chlorophyll and other particles.The extract was standardized in accordance with the method for determining the relative density (Bulgarian Pharmacopoeia Roll 2, p.19).The amount of extracted substances was on average 0.120 g/cm 3 .

Animals
Fifteen male New Zealand rabbits, purchased from the Animal House of the Agricultural Faculty, Trakia University, 5 months old, weighing 3.5-4.0kg, were used for this study.The experimental protocol was approved by the Department of Animal Care and adhered to the European Community Guiding Principles for the Care and Use of Animals.The animals were fed on a standard rabbit diet, had access to water ad libitum, and were synchronized by main-taining controlled environmental conditions (light, temperature, feeding time, etc.) for at least two weeks prior to and throughout the experiments.
The animals from group B were injected (IM) with HRE 2 h before irradiation.The animals from group C were injected (IM) with HRE 30 min after irradiation.All animals were irradiated with 60 Co gamma rays at dose 2.0 Gy.Blood samples were collected before treatment and 24 h, 3, 8 and 15 days after irradiation from the marginal ear vein in sterile tubes with EDTA.

Irradiation
A cobalt teletherapy unit (Rocus M, 60 Co) at the Inter-District Cancer Dispensary, Stara Zagora, Bulgaria, was used for irradiation.Each rabbit, in a wooden container, was exposed to 2.0 Gy gamma rays, at a dose rate 89.18 cGy/min.

Determination of oxidant/antioxidant balance
EDTA blood samples were centrifuged (1500 g, 15 min, 4°C) and plasma was carefully harvested and stored at -20°C until analysis.Erythrocyte lysate was prepared by the method of Ivanov (1999).
Superoxide dismutase (SOD) activity in the erythrocyte lysate was determined as described by Sun et al. (1988) with some modifications.The xanthine/xanthine-oxidase system was used for superoxide anion production.This anion reduces nitro blue tetrazolium (NBT) to formasan, which was monitored at 560 nm.The enzyme present in samples removes the superoxide anion and suppresses the reduction.The reduction rate was used to measure the SOD activity.One unit of SOD activity was determined as the enzyme quantity that causes 50% suppression of NBT reduction to formasan.Catalase (CAT) activity was estimated in the erythrocyte lysate by the method of Beers and Sizer (1952).The hemoglobin concentration of the lysate was determined by the cyanmethemoglobin method of Mahoney et al. (1993).
For the determination of the lipid peroxidation products, the method of Plaser and Cushman (1966), which measures MDA-reactive products, was used.

Statistical analysis
The results were expressed as mean ± standard deviation (SD) for five animals in each group.Differences between groups were assessed by one-way analysis of variance (ANOVA) using the SPSS 13.0 software package for Windows.Post hoc testing was performed for inter-group comparison using the least significance difference (LSD) test.P-values <0.05 was considered as significant.

RESULTS
Gamma irradiation caused a significant decrease in SOD activity (from about 53% at 24 h (925±226 U/gHb) to about 35% on day 15 of the experiment (1277±151 U/gHb) compared to the control (before treatment).However, administration of HRE both before and after irradiation caused an increase in the examined enzyme activity throughout the experiment.In the group treated with HRE before irradiation, SOD activity at 24 h (1466±158 U/gHb) was 58% higher, at 72 h (1616±169 U/gHb) 53% higher and on the 15 th day (1717±151 U/gHb) 34% higher than that in the irradiated group (2.0 Gy).Administration of HRE after irradiation caused an elevation of SOD activity, but the effect was less pronounced.(Fig. 1).
Whole-body gamma irradiation caused a significant increase in the level of the final lipid peroxidation product -malondialdehyde (MDA) at 24 h (5.41±0.38 µmol/L), 72 h (5.9±0.41 µmol/L),  day 8 (5.77±0.66µmol/L) and on day 15 (5.36±0.36µmol/L) by about 27, 39, 35 and 26%, respectively, in comparison to the control (before treatment).However, administration of HRE caused a significant decrease in the MDA levels during the whole time of the experiment.On the 15 th day, the decrease in levels was about 29% in the group treated with HRE before irradiation and 21% in the group treated with HRE after irradiation (Fig. 3).
The results indicate that administration of HRE both before and after irradiation could enhance antioxidative function, thus providing protection against a radiation-induced decrease in antioxidative ability and increase in lipid peroxidation.

DISCUSSION
Earlier we showed that HRE provides protection against radiation-induced chromosome aberrations in rabbit lymphocytes in vitro (Popov et al., 2010).In this study, we demonstrate that HRE can promote the antioxidative ability in 2.0 Gy of γ-ray-irradiated rabbits.
O 2 is reduced to H 2 O after receiving four electrons under the normal physiological condition, but partial reduction of O 2 induces the highly reactive oxygen species (ROS), including the superoxide anion (O 2 -), hydrogen peroxide (H 2 O 2 ), and the hydroxyl radical (OH).ROS can attack bioactive molecules such as DNA, protein and lipids, and damage their structures and functions, leading to aberrant downstream signaling or stimulation of apoptosis (Finkel, 1998;Thannickal et al, 2000).SOD, CAT and GSH-Px are important antioxidative enzymes that scavenge free radicals, thereby protecting cells from damage and decreasing deleterious species or lipid peroxides, such as MDA (Curello et al., 1987;Anscher et al., 2005).
In this study, the activity of the antioxidant enzymes SOD and CAT increased when rabbits were treated with HRE (Figs. 1 and 2).This suggests that HRE has a strong antioxidative effect.
MDA is an important oxidative metabolite of the polyunsaturated fatty acids that are found in biomembranes (Buege et al., 1978).MDA is often seen as an indicator of the oxidation status in cells or tissues.Thus, a high level of MDA is detrimental to cells and tissues, and leads to loss of their normal biofunction (Cheng et al., 2011).In this study, MDA content in the HRE-treated groups decreased signifi- cantly when compared with the control group (Fig. 3).These results suggest that HRE at the applied dose has a strong antioxidative capability.This is in agreement with the studies of Berkov et al. (2011) and Mihailova et al. (2011), who reported that HRE has a strong antioxidant and radical-scavenging activity.
Several pathways have been suggested for the mechanism of protective action in mammalian cells against the damaging effects of ionizing radiation (Weiss and Landauer, 2000).The mechanisms implicated in the protection of cells by radioprotectors include free radical scavenging that protects against reactive oxygen species (ROS) generated by ionizing radiation or chemotherapeutic agents, and hydrogen atom donation to facilitate direct chemical repair at sites of DNA damage (Bump and Brown, 1990).Shimoi et al. (1996) concluded that plant flavonoids that show antioxidant activity in vitro also function as antioxidants in vivo, and their radioprotective effect may be attributed to their radical-scavenging activity.ROS generated by ionizing radiation are scavenged by radioprotectors before they can interact with biochemical molecules, thus reducing the harmful effects of radiation.The antioxidant mechanism of radioprotection and free radical scavenging has been suggested to be a likely mechanism of radiation protection by the flavonoids orientin and vicenin (Uma Devi et al., 2000).
In this study, the administration of HRE (0.24 g/kg body weight) both before and after irradiation enhanced the activities of SOD and CAT when compared with the irradiated group only, but MDA content was significantly decreased.

CONCLUSION
The data presented here clearly indicate that HRE had a radioprotective effect against radiation-induced antioxidant damage by scavenging the excessive free radicals and lipid peroxides resulting from irradiation.However, the specific mechanism of HRE in enhancing antioxidant activity needs further investigation.

Fig. 1 .
Fig.1.Dynamics of SOD activity in the time interval before irradiation up to the 15 th day post-irradiation with a dose of 2.0 Gy in: Irradiated at 2.0 Gy group; Treated with HRE (0.24 g/kg b.w.) + irradiation 2.0 Gy group; Irradiated at 2.0 Gy + treatment with HRE (0.24 g/kg b.w.) group.Data are presented as mean ±SD; n=5;# statistical significance in comparison with control (before treatment); * statistical significance in comparison with irradiated group at the same day.

Fig. 2 .
Fig. 2. Dynamics of CAT activity in the time interval before irradiation up to the 15 th day post-irradiation with a dose of 2.0 Gy in: Irradiated at 2.0 Gy group; Treated with HRE (0.24 g/kg b.w.) + irradiation 2.0 Gy group; Irradiated at 2.0 Gy + treatment with HRE (0.24 g/kg b.w.) group.Data are presented as mean ±SD; n=5; # statistical significance in comparison with control (before treatment); * statistical significance in comparison with irradiated group at the same day.

Fig. 3 .
Fig.3.Dynamics of the plasma concentration of MDA in the time interval before irradiation up to the 15 th day post-irradiation with a dose of 2.0 Gy in: Irradiated at 2.0 Gy group; Treated with HRE (0.24 g/kg b.w.) + irradiation 2.0 Gy group; Irradiated at 2.0 Gy + treatment with HRE (0.24 g/kg b.w.) group.Data are presented as mean ±SD; n=5;# statistical significance in comparison with control (before treatment); * statistical significance in comparison with irradiated group at the same day.