The focus of this article is an evaluation of in vitro antioxidant activity and cytotoxicity of Rosa damascena extract using peripheral blood lymphocytes in a model system. Rosa damascena today is highly cultivated all over the world for its visual beauty and scent.
In addition to its perfuming effect, flowers, petals and hips (seed pods) of Rosa damascena are used for medical purposes. There is an increasing interest in the natural antioxidants present in medicinal plants, which are candidates for the prevention of oxidative damage that has been proposed as one of the underlying molecular mechanism for the development of various diseases such as cancer, inflammation, and coronary heart disease.1 Although previous in vitro and in vivo studies have demonstrated that various Rosa species cause an improvement in antioxidant activities of various tissues and act to limit oxidative damage,2-6 little information is available about the effect of the Rosa damascena on these actions. Therefore, the aim of the present study is to investigate the in vitro ability of the aqueous extract from the Rosa damascena flower to protect against oxidative stress induced cytotoxicity in peripheral blood lymphocytes obtained from healthy subjects, and to compare it with the activity of N-acetylcysteine (NAC), a known antioxidant and free radical scavenger. In addition, as the antioxidant substances may also exert pro-oxidant effects, it is important to investigate the concentration dependent effects thereof. Therefore, concentration-dependent effects of the aqueous extract were also evaluated in lymphocytes.
Materials and methods
Preparation of rose flower extract Dried rose flowers from mainland Turkey (Denizli-Baçmakçi province, Baçmakçi Rose Cooperative), collected in June 2007, were macerated in distilled water for 24 hours and then boiled under reflux for 4 hours. After cooling, the extract was filtered using filter paper, and the filtrate was lyophilised. The resulting powder was used in the study.
Lymphocyte preparation, cell culture and treatment
Following informed consent, lymphocytes were isolated from heparinised whole blood of normal healthy donors by standard gradient centrifugation (15 min, 300 g) using Histopaque 1077. Lymphocytes were harvested from the interface, then washed once with PBS and once with RPMI-1640 by centrifugation at 400 g for 5 min. Cell viability was measured by use of the Trypan-blue exclusion assay. Approximately 1 x 106 cells were seeded in RPMI medium containing L-glutamine (2 mM), fetal calf serum (20%), penicillin (100 UI/ml) and streptomycin (100 (μg/ml) and incubated at 37°C. Lymphocyte stimulation was performed by addition of phytohaemagglutinin (1.5%). At 1 hour, various concentrations of rose extract or NAC were added to cultures from each individual. After pre-incubation with Rosa extract or NAC for 2 hours, oxidative stress was induced by addition of cumene hydroperoxide (CumOOH) and then all cell suspensions were incubated at 37°C for 24-72 hours.
Determination of luminal-dependent chemiluminescence
The measurement of luminol-dependent chemiluminescence (CL) was similar to that described previously.7 After cells were exposed to agents, the cell suspensions were collected in a polystyrene cuvette and were incubated with 10 μl of 0.3 M NADPH at 37°C for 15 minutes. The chemiluminescence was then measured in an absolutely dark chamber of the luminometer. At the 100-s timepoint, 10 μL of 5 mM luminol in phosphate buffered saline (PBS) was added into the cell suspensions. The chemiluminescence in the sample was continuously measured in a luminometer (Luminoskan TL luminometer, Labsystems Inc., Helsinki, Finland) for a total of 5 minutes. Negative controls were prepared by adding 10 μL of luminol, and 10 μl NADPH to 400 μl PBS. The assay was performed in duplicate for each sample and the CL level was monitored as relative light units. Chemiluminescence inhibition is reported as the ratio of the maximum emission in the presence of plant extracts to that in their absence (control). The antioxidant activity (AA) of the samples was calculated from the equation: AA (%) = ((IO -I)/IO) x 100, where IO and I, are the relative light intensities of the blank and sample solutions, respectively. Setting IO/I = 2, it was possible to calculate the amount of the rose extract needed to provoke 50% reduction in ICL (IC50).
Evaluation of intracellular cell oxidants
The cellular redox state was analysed by determination of the oxidation of DCFH to the fluorescent 2’, 7’-dichloroflurecent (DCF). After the lymphocyte culture with Rosa damascena and/or CumOOH, the cells were washed in ice-cold PBS and were added to 1 ml of PBS containing 20 μM DCFH-DA.8 Then the cells were incubated for 30 min at 37°C. The DCF fluorescence emission was then analysed using a Shimadzu RF-5000 spectrofluorophotometer with the excitation and emission spectra set at 480 and 530 nm (with slit widths of 5 nm), respectively.
Measurement of total cellular glutathione (GSH)
Before incubation with individual agents, lymphocytes were washed and pelleted at 500 g for 10 minutes. Total GSH analysis was performed using a modification (Griffin)9 of the method of Tietze.10
Hoechst 33258/propidium iodine staining assay
The percentage of changed (apoptotic and necrotic) cells was evaluated by fluorescence using Hoechst 33258/ propidium iodine as described by Zhang et al.11
Statistical analysis
Each experiment (n ³ 5) was run at least in duplicate and the data presented are given as mean ± SD. Statistical analysis of data was performed by analysis of variance (ANOVA) using the SPSS 16 for Windows (SPSS, Chicago, IL). P<0.05 was considered statistically significant for all experiments.
Results and discussion
The aqueous extracts from the Rosa damascena flower were primarily evaluated for antioxidant activity using a luminoldependent chemiluminescence assay. In the present study, in vitro effects of Rosa damascena were compared with those of NAC, commonly used by the pharmaceutical industry. To this end, the lymphocytes were pre-treated for 2 hours with the extract prior to an induction of oxidative stress with CumOOH, a non-polar oxidising agent used in industry, for 24-72 hours. It is easily taken up by cells and is not metabolised by catalase. As parameters for radical scavenging activity, the IC50 value (i.e., the sample concentration causing 50% inhibition of luminol-enhanced chemiluminescence) was used. Exposure to CumOOH alone resulted in a dramatic increase in luminolenhanced chemiluminescence intensity (data not shown). As shown in Table 1, all Rosa damascena concentrations tested in this study exhibited considerable inhibitory activity against CumOOH-induced oxidative stress, although high concentrations (> 500 μg/ml) increased the CumOOHinduced chemiluminescence formation. The IC50 for Rosa damascena was 19 (g/mL, whereas NAC showed IC50 of 54 μg /mL. in this methodology. Under the same conditions, Rosa damascena extract at concentrations near the IC50 values decreased the oxidation of DCFH by CumOOH (>60% of reduction) compared to NAC. The oxidation of DCFH originates DCF, which is a fluorescent compound. Accumulation of DCF indicates the production of redox-active substances in the cell and DCF fluorescence levels reflect the intracellular concentration of ROS. These results suggest that the extract of Rosa damascena acts to inhibit the oxidative stress in a more effective way than NAC. GSH levels were also measured after the 24-hour combined exposure to 15 μM CumOOH and 20 μg/ml Rosa damascena in cells previously incubated with Rosa damascena extract for 24 hours or not. Treatment with CumOOH significantly decreased the intracellular GSH content, whereas pre-incubation of cells with Rosa damascena extract prevented this effect (Table 2). Because other rose species are believed to have a wide range of other pharmaceutical activities including anticytotoxic and anti-genotoxic effect against radical induced agents, the rates of apoptosis and necrosis in peripheral blood lymphocytes after exposure to Rosa damascena alone (20 μg/ml) and in combination with CumOOH were also evaluated using Hoechst 33258 and propidium iodine. As shown in Table 3, CumOOH decreased the viability of the lymphocytes. Cells in early and late apoptosis as well as necrosis were also observed. However, pretreatment with Rosa damascena extract increased the number of living cells and decreased the apoptotic and necrotic cell numbers. Similar effects were also found with NAC. Various Rosa species have been reported to prevent the increase of free radical scavengers in lipid peroxides. Our findings support previous findings regarding the antioxidant effects of various Rosa flowers.2-6
Conclusions
In conclusion, our results suggest that the aqueous extract of the Rosa damascena flower in concentrations of about 20 μg/ml has antioxidant properties against CumOOH-induced oxidative stress and cytotoxicity under the experimental conditions tested. It is concluded that these results support the associated health-promoting potential of the Rosa damascena flower, in particular against oxidative stress.
Acknowledgement
This work was supported by BIOTA Herbal Cosmetic Laboratories Ltd. Co.
References 1 Inagi R. Recent Patents on Cardiovascular Drug Discovery. Bentham Science, 2006. 151-159. 2 van der Westhuizen F.H., van Rensburg C.S., Rautenbach G.S., Marnewick J.L., Loots du T., Huysamen C., Louw R., Pretorius P.J., Erasmus E. Phytother. Res. 2008. 22 (3): 376-383. 3 Franco D., Pinelo M., Sineiro J., Nú˜nez M.J. Bioresour Technol. 2007. 98 (18): 3506-3512. 4 Ng T.B., Pi Z.F., Yue H., Zhao L., Fu M., Li L., Hou J., Shi L.S., Chen R.R., Jiang Y., Liu F. J Pharm. Pharmacol. 2006. 58 (4): 529-534. 5 Sharma S., Sultana S. Basic Clin. Pharmacol. Toxicol. 2004. 95 (5): 220-225. 6 Daels-Rakotoarison D.A., Gressier B., Trotin F., Brunet C., Luyckx M., Dine T., Bailleul F., Cazin M., Cazin J.C. Phytother. Res. 2002. 16 (2): 157-161. 7 Sun J.S., Hang Y.S., Huang I.H., Lu F.J. Free Radical Biol Med. 1996. 20: 107-112. 8 LeBel C.P., Ischiropoulos H., Bondy S.C. Chem. Res. Toxicol. 1992. 5: 227-231. 9 Griffin O.W. Anal. Biochem. 1980. 106: 207-212. 10 Tietze F. Anal. Biochem. 1969. 27: 502-522. 11 Zhang L., Mizumoto K., Sato N., Ogawa T., Kusumato M., Niiyama H., Tanaka M. Cancer Lett. 1999. 142: 129-137.
