Increasing evidence suggests that free radical-induced oxidative damages lead to various pathological events including coronary heart disease, cancer and aging . In p a r t i c u l a r, lipid peroxidation in biological membranes has attracted much attention in relation to the deterioration of membrane structure and impairment of enzymatic functions .
The oxidation of erythrocyte membranes serves as a model for the oxidative damage of biomembranes [3, 4]. The oxidation of erythrocyte and its ghost’s membranes induced by free radicals have also been studied, and it has been found that free radicals generated in the aqueous phase attack the membrane to induce the chain oxidations of lipids and proteins and eventually cause hemolysis [5, 6]. Much interest exists in the possibility that antioxidants reduce the risk of degenerative diseases by inhibiting free radical induced oxidative damage . Therefore, several studies have examined both natural and synthetic antioxidants for the inhibition of lipid peroxidation in membrane systems.
Rosemarinus officinalis L. (Labiatae) is a common household plant grown in many partsof the world . Its extracts have been widely used as preservative in food industry due to the antioxidant activity of some of its constituents such as carnosol and carnosoic acid . These compounds have been shown to inhibit the non enzymatic-induced lipid peroxidation in liver microsomes when incubated in the presence of FeCl3 . In isolated rat hepatocytes, an aqueous extract of R. officinalis has been shown to reduce tert-butylhydroperoxide effects on lipid peroxidation .
Based on these precedents, it can be inferred that blocking free radical propagation and lipid peroxidation would protect erythrocytes against the deleterious eff e c t s of oxidative stress. In the present study, erythrocytes were oxidized by a water-soluble radical generator 2,2’ azobis (2-amidinopropane) hydrochloride (AAPH) and the protective effect of rosemary essential oil on hemolysis was investigated.
Figure 1.Effect of different concentrations of rosemary essential oil and AAPH on erythrocytes (n= 5, mean ± SEM, ***p < 0.001 compared to PBS).
2. Materials and Methods
2.1. Plant material and essential oil isolation
The aerial parts of R. officinalis L. were collected in Mashhad, Iran, dried in shade, and then grounded. The plant was identified by the Department of Botany, Ferdowsi University, Mashhad, Iran. A voucher specimen was deposited in the herbarium of the Faculty of Pharmacy, Mashhad (153-1815-06).
The oil was obtained by hydrodistillation using a clevenger-type apparatus for 3 h to give pale yellow oil. It was dried over anhydrous sodium sulfate and stored at 4-8 ºC in the dark. The yield of the extract was 1.5% (v/w).
2.2. Experimental procedure
The method is based on the modification of the method explained by Jimenez et al. . Briefly, blood was obtained from ether-anaesthetized rats and quickly centrifuged (3000 rpm, 10 min, 22 ºC). The erythrocytes were washed three times with saline (0.9%) and centrifuged (3000 rpm, 10 min) to obtain a constantly packed cell volume. The latter was subsequently used to prepare a 10% v/v erythrocyte suspension with phosphate buffer (pH=7.4). Aliquots (2.5 ml) of the cell suspension were added to 25 ml flasks and incubated at 37 ºC for 10 min with constant and gentle shaking. Two final concentrations of AAPH (25 and 50 mM) which prepared in phosphate buffer and four final concentrations of essential oil (0.178, 0.357, 0.535 and 0.714 ml/ml) were added to the incubation medium. Samples of the suspensions were removed at different time points and the degree of hemolysis was assessed spectrophotometrical-ly by reading the absorption of supernatant at 540 nm (absorption A). Similarly, the reaction mixture was treated with 0.1% triton X-100 solution to yield complete hemolysis, and the absorption of the supernatant after centrifugation was measured at 540 nm (absorption B). The percentage of hemolysis was calculated from the ratio of readings, (absorption A/ absorption B) x100.
Figure 2. Protective effect of different concentrations of rosemary essential oil on the AAPH (25 mM)– induced erythrocytes lysis (n=5, mean±SEM, *** p < 0.001 compared to PBS).
2.3. Statistical analysis
Data were presented as mean±SEM. All data were analyzed using analysis of variance (ANOVA) followed by tukey-kramer test for comparison. Statistical significance was defined as p < 0.05.
Erythrocytes suspended in phosphate b u ffer solution and incubated at 37 ºC remained stable and little hemolysis was observed for 6 h (less than 8%). The addition of different concentrations of the essential oil to erythrocytes suspension showed no significant hemolysis compared with phosphate buffer solution. When AAPH (25 mM and 50 mM) was added to erythrocytes suspension, it induced hemolysis in a time-dependent manner and was characterized by an induction phase, during which no significant hemolysis took place, followed by a sharp hemolysis phase (Figure 1). The essential oil inhibited hemolysis induced by 25 mM AAPH in a concentration-dependent manner. However, in the presence of 50 mM of AAPH, only the two higher concentrations of the essential oil (0.535 ml/ml and 0.714 ml/ml) inhibited hemolysis (Figures 2 and 3).
The effects of delayed addition of the essential oil on AAPH-induced erythrocytes lysis are shown in Figure 4. The addition of essential oil 2 h or 3 h after incubation with AAPH (25 mM) had no significant effect on the time course of cell lysis.
Figure 3. Protective effect of different concentrations of rosemary essential oil on the AAPH (50 mM)– induced erythrocytes lysis (n=5, mean±SEM, *** p < 0.001 compared to PBS).
Data presented here show that, in addition to its demonstrated antioxidant eff e c t s , rosemary essential oil also displays cytoprotective effects against the damage induced by AAPH to the intact erythrocytes. As rosemary has the ability to scavenge peroxyl radicals , its cytoprotective effects are likely to result from diminishing AAPH-derived peroxyl radicals generated during the incubation period. It is possible that during the induction period, antioxidant molecules in the essential oil are progressively consumed by AAPH–derived peroxyl radicals. Such scavenging action would lower the extracellular concentration of peroxyl radicals to a level that could be accounted for the reduction in the maximal extent of the hemolysis seen when the essential oil is added to the cell suspension.
Recent studies by Sato et al.  have shown that the hemolytic action of A A P H could be associated with the oxidation of lipid components present in the erythrocyte membranes. Thus, the possibility that rosemary exerts its cytoprotective effects at the membrane level, as a chain-breaking antioxidant, should also be considered. In fact, Miki and Mino  established that under experimental conditions similar to those employed here, the hemolysis ensues only after AAPH has lowered the erythrocyte membrane α-tocopherol concentrations to a critically low level. Therefore, rosemary essential oil may interact with lipoperoxyl radicals and spare α-tocopherol molecules. Membrane protein degradation is a major event associated with the A A P H - i n d u c e d lysis of erythrocytes . On the other hand, rosemary may protect red blood cells lysis by preventing the oxidative damage of membrane proteins.
Figure 4. Effect of delayed addition of rosemary essential oil on the AAPH (25 mM)–induced erythrocytes lysis (n=5,mean±SEM, *** p < 0.001 compared to PBS).
Our study also showed that delaying the addition of the essential oil to the incubation mixture for 2 to 3 h resulted in the loss of the effectiveness of essential oil to prevent lysis induced by AAPH. This result suggests that within this elapsed time, AAPH has already triggered oxidative modifications that can no longer be prevented or reversed by the presence of rosemary essential oil.
Since the leakage of hemoglobin into the extracellular medium represents only a final event to measure the extent of cell lysis, it is not possible from the present data to determine the mechanism(s) by which essential oil exerted its cytoprotective effects. However, given the possibility that the oxidation of membrane lipids or proteins represents a significant event underlying erythrocyte lysis, future work will address whether rosemary prevents the oxidation of both membrane components and how such effects would relate to its reported cytoprotective actions.
The authors are thankful to the Vi c e Chancellor of Research, Mashhad University of Medical sciences for financial support.