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Background and Objectives: There is a profound inclination among people toward consumption of herbs and herbal products. Some of these products are harmful while health promoting potentials of some others should be discovered. In the present study the antibacterial, antioxidant, acute and subchronic and cancer cell toxicity of methanolic and aqueous extracts of Rosa damascena Mill. were studied.
Material and Methods: Antimicrobial activities were determined by agar disc diffusion method. Total phenol content was estimated. Antioxidative properties of the extracts were determined by bleaching of beta carotene or 2,20-diphenylpicrylhydrazyl (DPPH). The Ferric-Reducing Antioxidant Power (FRAP) was expressed as gallic acid equivalents or known Fe(II) concentration for rose extracts and blood sera respectively. Acute and subchronic toxicity and cytotoxicity of the extracts were tested using animal model or Hela cells. Hematology and clinical chemistry parameters were noted.
Results: S.aureus was found susceptible. The total phenol contents of the methanolic and aqueous extracts were 132.67±3.51 and 117.33±6.81 μg Gallic acid equivalent/mg sample respectively. Antioxidative effects were higher than those of the synthetic antioxidants. A dose dependent levels of FRAP was noted in blood sera of rats gavaged with the extracts. Decrease in cholesterol/HDL and LDL/HDL ratios, fasting glucose, blood urea nitrogen, creatinine and uric acid is suggestive of promising therapeutic potentials of the extract. Inhibitory concentration of 50% (IC50) of 4.5 µg/ml was determined for cytotoxicity of the extract against Hela cell line.
Conclusion: The results suggest application of rose extract as a natural antioxidant and health promoting agent.
Key words: Rose, Antimicrobial Agents, Antioxidant; Cytotoxicity
Received: 22 December 2009
Address communicated to: Dr Iraj Rasooli, Department of Biology, Shahed University, Tehran,
Email: [email protected]
Herbs and herbal extracts contain different phytochemicals with biological properties that promote human health and help reduce the risk of chronic disease (1). Over 150 rose species and more than 2000 cultivars have been registered (2). Members of the Rosaceae family have long been used for food and medicinal purposes. Fresh (FF) and spent (SF) Rosa damascena flower extracts were effective against all the bacteria except E. coli O157:H7. FF and SF extracts showed the strongest effects against S. enteritidis and M. smegmatis, respectively (3). Antimicrobial activity of Rosa damascena was recorded only to Staphylococcus aureus (4). Total aerobic mesophilic bacteria, Enterococci, Enterobactericeae and Staphylococcus aureus counts were decreased in the dried rose dreg group as an alternative litter material for broiler performance and microbiological characteristics of litter at 42 days. The physiological functions of Rosaceae may be partly attributed to their abundance of phenolics. Phenolic acids and flavonoids, known as bioactive agents, frequently occur in herbal plants (5). The correlation coefficients between antioxidant activity, on the one hand, and the contents of total phenols and of gallic acid in various rose cultivars, on the other hand, were 0.79 and 0.81, respectively. It was concluded that dried rose petals may be used for preparing antioxidant-rich caffeine-free beverages, either separately or in combination with other herbal materials (6). Phenolics possess a wide spectrum of biochemical activities, such as antioxidants, free-radical scavengers (7), anticancer (8) and anti-inflammatory (9); however the antioxidative properties remain the core topic of investigation in recent years. Crude extracts of the plant parts rich in phenolics are increasingly of interest in the field of nutrition, health and medicine, because they retard oxidative degradation of lipids and thereby improve the quality and nutritive value of foods (10). Supplementing Drosophila with Rosa damascena extract resulted in a statistically significant decrease in mortality rate in male and female flies. Moreover, the observed anti-aging effects were not associated with common confounds of anti-aging properties, such as a decrease in fecundity or metabolic rate (11). However, no report till date was available on the characterization of phenolic constituents in wild growing R. damascena until recently reported an RP-HPLC method for the determination of polyphenols in R. damascena, R. bourboniana and R. brunonii (12). The aim of this study was to determine the health promoting potentials of Rosa damascena Mill. extracts grown extensively and consumed widely in Iran. Thus, herein we report the assessment of antimicrobial, antioxidant capacities, total phenolics, acute and subchronic toxicity, and human cell anti tumor cytotoxicity of Rosa damascena Mill. extracts.
Materials and Methods
Equipments and chemicals
The major equipments used were, UV-2501PC spectrophotometer, ELISA reader and routine microbiology laboratory equipments. Microbial and cell culture media and laboratory reagents were from Merck, Germany. Other chemicals were of analytical grade.
Preparation of extracts
Rosa damascena collected from the natural rose gardens of Kashan city of Iran were shadow dried. The dried flowers were ground finely. Aqueous extract was prepared by adding 100 g of the powder to 500 ml of boiling water for 30 minutes. After filtration, the extract was lyophilized with a freeze-dryer and stored at 4°C. 500 ml of methanol was used for methanolic extraction at room temperature for 3 h. After extraction, the mixture was filtered and the residue was re-extracted with 500 ml of fresh methanol overnight. The combined methanolic solution was centrifuged at 12,000g for 10 min. The extracts were distilled under vacuum at 40 °C, dried in lyophilizer and stored at 4 °C until use. The methanolic extract was reconstituted in dimethyl sulfoxide (DMSO) to a concentration of 400 mg/ml for subsequent experimentation.
Microbial strain and growth media
E. coli (ATCC 25922), S. aureus (ATCC 25923), Streptococcus faecalis (PTCC 33186), Pseudomonas aeruginosa (ATCC 8830) and Klebsiella pneumoniae (ATCC 13883) were employed in the study. Bacterial suspensions were made in Brain Heart Infusion (BHI) broth to a concentration of approximately 108 cfu/ml. The suspension concentrations were measured spectrophotometrically. Subsequent dilutions were made from the above suspension, which were then used in the tests.
Extract sterility test
In order to ensure sterility of the extracts, geometric dilutions ranging from 0.04 to 80 mg/ml of the extracts, were prepared in a 96-well microtitre plate, including one growth control (BHI+DMSO) and one sterility control (BHI+DMSO+test extract). Plates were incubated under normal atmospheric conditions, at 37oC for 24 h. The contaminating bacterial growth, if at all, was indicated by the presence of a white ''pellet'' on the well bottom. The extracts were filter sterilized, as and when needed, using 0.45µ sterile filter (13).
Disc diffusion method
The agar disc diffusion method (13) was employed for the determination of antimicrobial activities of the extracts in question. Briefly, 0.1 ml from 108 CFU/mL bacterial suspension was spread on the Mueller Hinton Agar (MHA) plates. The agar was bored with a sterile borer (6 mm in diameter). 50μl of the 20mg/ml and 10mg/ml dilutions of each extract were placed in the wells of the inoculated plates. The plates were allowed to stand for 1 hour at room temperature, then at 4oC for 2h. The plates were then incubated at 37oC for 24 h. The diameters of the inhibition zones were measured in millimeters. All tests were performed in triplicate.
Total phenolic content assay
Total phenol content was estimated as gallic acid equivalents (GAE; mg gallic acid/g extract) as described earlier (14). In brief, a 100 μl aliquot of dissolved extract was transferred to a volumetric flask, containing 46.0 ml distilled H2O, to which was subsequently added 1 ml Folin-Ciocalteu reagent. After 3 mins, 3 ml of 2% Na2CO3 was added. After 2 h of incubation at 25°C, the absorbance was measured at 760 nm. Gallic acid (Sigma Co., 0.2-1 mg/ml gallic acid) was used as the standard for the calibration curve, and the total phenolic contents were expressed as mg gallic acid equivalents per gram of tested extracts (Y=0.001x +0.0079; r2 = 0.9967).
DPPH Radical Scavenging Capacity of the Extracts
The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-colored methanol solution of 2,20-diphenylpicrylhydrazyl (DPPH). Two ml of different dilutions of the extract in methanol were added to two ml of a 0.0094% methanol solution of DPPH. Trolox (1 mM) (Sigma-Aldrich), a stable antioxidant, was used as a synthetic reference. After a 30 min incubation period at room temperature, the absorbance was read against a blank at 517 nm. Inhibition of free radical by DPPH in percent (I%) was calculated in following way (13):
I% = (Ablank â”€ Asample/Ablank) - 100;
where Ablank is the absorbance of the control reaction (containing all reagents except the test compound), and Asample is the absorbance of the test compound. Tests were carried out in triplicate.
Lipid peroxidation inhibition activity
Lipid peroxidation inhibition activity was determined using the β-carotene bleaching assay. Approximately 5 mg of β-carotene (type I synthetic, Sigma-Aldrich) was dissolved in 10 ml of chloroform. The carotene-chloroform solution, 1.5 ml, was pipetted into a boiling flask containing 33.82 mg linoleic acid (Sigma-Aldrich) and 300 mg Tween 40 (Sigma- Aldrich). Chloroform was removed using a rotary evaporator at 40oC for 5 min and, to the residue, 150 ml of distilled water were added, slowly with vigorous agitation, to form an emulsion. 2.5 ml of the emulsion were added to a tube containing 350 μl of the test extract dilutions and the absorbance was immediately measured at 470 nm against a blank, consisting of an emulsion without β-carotene. The tubes were placed in a water bath at 50oC and the oxidation of the emulsion was monitored spectrophotometrically by measuring absorbance at 470 nm over 30, 60 and 90 minute periods. Control samples contained 350 μl of water instead of the test extract. Butylated hydroxy anisole (BHA) and butylated hydroxytoluene (BHT), stable antioxidants, were used as synthetic references. Lipid peroxidation inhibition activity was expressed as percent antioxidant activity AOA (%) and calculated as follows (13):
Bleaching rate (BR) of β-carotene= ln(Ainitial/ Asample)/time (minutes)
AOA (%)=1- (BRsample/ BRcontrol)-100
Where Ainitial and Asample are absorbance of emulsion before and after incubation period, and (BRsample and BRcontrol are bleaching rates of the sample and negative control respectively.
Ferric-Reducing Antioxidant Power (FRAP) Assay of the Extract
The FRAP assay was carried out according to the procedure employed by (15). One millilitre of the extract dilution was added to 2.5 ml of 0.2 M potassium phosphate buffer (pH 6.6) and 2.5 ml 1% potassium ferricyanide. The mixture was incubated for 20 min at 50 °C, after which 2.5 ml of 10% trichloroacetic acid was added. The mixture was then separated into aliquots of 2.5 ml and mixed with 2.5 ml of deionised water. Then, 0.5 ml of 0.1% (w/v) FeCl3 were added to each tube and allowed to stand for 30 min. Absorbance for each tube was measured at 700 nm. The FRAP was expressed as gallic acid equivalents (GAE) in mg/g of samples used (y = 16.667x+0.0038; r2 = 0.9991).
Serum Ferric Reducing Antioxidant Power (FRAP)
The antioxidant power of blood serum was determined using FRAP assay (16). Briefly, 50 μl of the blood serum (normal as well as experimental cells) suspension was added to 1.5 ml of freshly prepared and pre-warmed (37 oC) FRAP reagent (300 mM acetate buffer, pH = 3.6, 10 mM TPTZ (tripyridyl-s-triazine) in 40 mM HCl and 20 mM FeCl3.6H2O in the ratio of 10:1:1) and incubated at 37 oC for 10 min. The absorbance of the sample was read against reagent blank (1.5 ml FRAP reagent + 50 μl distilled water) at 593 nm. Aqueous solutions of known Fe(II) concentration (FeSO4.7H2O) were used for calibration of the FRAP assay and antioxidant power was expressed as µg/ml (y = 0.0025x+0.0005; r2 = 0.9976).
Acute and subchronic toxicity
In order to avoid any toxic effect of residual methanol in the extract and with respect to almost equal antioxidative properties of both extracts, this and cytotoxicity parts of the study were performed with aqueous extract of R. damascene only. A 30-day oral toxicity study was conducted in Wistar rats (Rattus norvegicus; 180-200 g) to determine the potential of R. damascena methanolic extract to produce toxic effects. The rats of both sexes, were housed in temperature-controlled rooms and were given food and water ad libitum until used. The test extract was administered via oral gavage to the rats (n = 10 mice per group) orally at doses of 2.5, 5, 25 and 50 mg/kg/day corresponding approximately to doses of 0.5, 1, 5 and mg/animal/day respectively. The results obtained were compared with those for the control animals [0.9% saline]. The LD50 was calculated by the probit method by using SPSS 7.0 for Windows. To investigate the subchronic toxicity of the rose extract, after 30 days of oral administration to rats, the haematological and serum biochemistry parameters were evaluated. Blood samples were collected by puncture in the infraorbital plexus. The blood samples collected on day 0 and day 30 were used for determining red cell and leucocyte counts and for haemoglobin, haematocrit and biochemical parameter analysis. The serum concentrations of urea, creatinine, glutamic-oxalacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) and other parameters were determined by using commercial kits. The values obtained were compared within and between the groups (17).
The human cervical carcinoma Hela cell line NCBI code No. 115 (ATCC number CCL-2) were procured from Pasteur Institute, Tehran-Iran. The cells were grown in RPMI 1640 supplemented with 10% fetal calf serum, 1% (w/v) glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured in a humidified atmosphere at 37 °C in 5% CO2. Cytotoxicity was measured using a modified MTT assay. This assay detects the reduction of MTT [3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide] by mitochondrial dehydrogenase, to blue formazan product, which reflects the normal functioning of mitochondrial and cell viability (17). Briefly, the cells (5 - 104) were seeded in each well containing 100μl of the RPMI medium supplemented with 10% FBS in a 96-well plate. After 24 h of adhesion, a serial of doubling dilution of the test extract was added to triplicate wells to the final concentration range of 5-0.1 mg/ml reaction well. The final concentration of ethanol in the culture medium was maintained at 0.5% (volume/volume) to avoid toxicity of the solvent (18). After 2 days, 10 μl of MTT (5 mg/ml stock solution) were added and the plates were incubated for an additional 4 h. The medium was discarded and the formazan blue, which formed in the cells, were dissolved with 100 μl dimethyl sulphoxide (DMSO). The optical density was measured at 490 nm using a microplate ELISA reader. The cell survival curves were calculated from cells incubated in the presence of 0.5% ethanol. Cytotoxicity is expressed as the concentration of drug inhibiting cell growth by 50% (IC50), (y = 2154.3x+40.22; r2 = 0.974). All tests and analyses were run in triplicate and mean values recorded.
All the experimental data are presented as mean ± SEM of three individual samples. Data are presented as percentage of free radical scavenging/inhibition lipid peroxidation on different concentration of Rosa damascena extract. IC50 (the concentration required to scavenge 50% of free radicals) value was calculated from the dose-response curves. Antibacterial effect was measured in terms of zone of inhibition to an accuracy of 0.1 mm and the effect was calculated as a mean of triplicate tests. All of the statistical analyses were performed with the level of significant difference between compared data sets being set at p < 0.05.
The antibacterial effect of Rosa damascena extracts were tested against some pathogens by agar diffusion and dilution methods. S. aureus was equally sensitive to both methanolic and aqueous extracts. E.coli, S.faecalis, Pseudomonas aeruginosa and Klebsiella pneumoniae were resistant (Table 1).
Table 1. Determination of growth inhibition zone of S. aureus exposed to Rosa damascena extracts, the total phenolics of the extracts and mean inhibition of DPPH free radical (%)
Extracts and synthetic antioxidants
Mean Inhibition Zone (mm) 50µl
Mean Inhibition Zone (mm) 50µl (0.5 mg)/Well
DPPH scavenging effect (%)
Total phenolic content GAE
μg Gallic acid/mg sample
Total Phenolics Content(TPC)
The total phenol contents (TPC) of the methanolic and aqueous extracts of R. damascena flower were determined to be 132.67±3.51 and 117.33±6.81 μg Gallic acid equivalent /mg sample (GAE/mg) respectively (Table 1).
The antioxidant capacities of the rose extracts as assessed by different assay methods are summarized in Tables 1-3. R. damascena extracts exhibited a dose-dependent scavenging of DPPH radicals and 49 μg/ml and 40 μg/ml of the methanolic and aqueous extracts were sufficient to scavenge 50% of DPPH radicals respectively (Table 1). In the peresnt study DPPH scavenging effect (%) of the extracts were significantly higher than those of the synthetic antioxidants (Table 1). Lipid peroxidation inhibition activity of Rosa damascena extracts determined by β-carotene bleaching assay revealed statistically equal potency to the standard BHT and BHA (Table 2). Ferric-reducing antioxidant power (FRAP) of the methanolic and aqueous extracts were determined as 103.9±7.86 mg/g and 97.6±3.3 mg/g respectively. The FRAP of the aqueous extracts tested in blood sera of the rats gavaged with a daily dose of 50, 25, 5 and 2.5mg/kg showed a dose dependent and increased levels of ferric-reducing antioxidant power as compared to the control group (Table 3).
Table 2. Lipid peroxidation inhibition activity of Rosa damascena extracts determined by β-carotene bleaching assay at different time intervals
Rosa damascena methanolic extract
Rosa damascena aqueous extract
Table 3. Serum Ferric-Reducing Antioxidant Power (FRAP) assay of Rosa damascena extracts
FeSO4.7H2O equivalent (μg/ml)
Test/Control Ratio (%)
Acute and subchronic toxicity
There were considerable treatment-related effects in hematology and clinical chemistry parameters (Table 4). There was increased body weight in test groups as compared to the control. However the percent weight gains were not statistically significant. Significant decrease in total white blood cell (WBC) was noted at highest and lowest concentrations of the extracts while platelet counts were significantly increased in all test groups. Fasting glucose, SGOT and SGPT levels were significantly decreased and alkaline phosphatase levels were significantly increased in all test groups (Table 4). Clinical chemistry parameters also showed increased levels of triglycerides. This increase was statistically significant only in high dose group. Interestingly, cholesterol/HDL ratio and LDL/HDL ratio were also higher in the sera of the high dose group while these levels were significantly decreased in other three doses of 25, 5 and 2.5 mg/kg/day groups. This, and decrease in other parameters such as fasting glucose, blood urea nitrogen (BUN), creatinine (CREA) and uric acid (Table 4) is suggestive of promising therapeutic potentials of R.damascena extract at lower doses.
Table 4. Mean hematology and clinical chemistry values of rats blood samples fed with Rosa damascena aqueous extract
Initial Body weight (g)
Final Body weight (g)
Weight gain (%)
Erythrocyte count (RBC) (-106/lL)
Total white blood cell (WBC) and differential leukocyte count (-103/µL)
Hemoglobin concentration (HGB) (g/dL)
Hematocrit (HCT) (%)
Platelet count (PLT) (-103/µL)
Red Cell Distribution Width [RDW (%)]
Mean Platelet Volume (MPV)
Mean corpuscular volume (MCV) (fL)
Mean corpuscular hemoglobin (MCH) (pg)
Mean corpuscular hemoglobin Concentration [MCHC (g/dL)]
Fasting glucose (GLUC) (mg/dL)
Blood Urea nitrogen (BUN) (mg/dL)
Blood creatinine (CREA) (mg/dL)
Total cholesterol(CHOL) (mg/dL)
Triglycerides (TRIG) (mg/dL)
Alkaline phosphatase (ALKP) (U/L)
The aqueous extract of R.damascena at 0.5 mg/ml concentration destructed cells by 74.11% (Table 5). The 50% cytotoxic concentration was found to be 4.5 µg/ml.
Table 5. Cytotoxicity assay of Rosa damascena aqueous extract
Extract Dilutions (mg/ml)
% Viable Hela cell line
25.89 ± 0.36
26.67 ± 0.48
28.20 ± 1.08
30.24 ± 1.02
38.74 ± 1.22
47.5 ± 0.78
55.4 ± 0.92
Susceptibility of S.aureus is consistent with those reported earlier (4). Both water and ethanolic extracts of Rosa damascena were effective on methicillin-resistant Staphylococcus aureus (MRSA). The ethanolic extract with the greatest antimicrobial activity was that of R. damascena (MIC 0.395 to 0.780 mg/ml and MBC 1.563 to 3.125 mg/ml) (19). The resistance of E.coli in the present study confirms report of other investigators (3). It is suggested that the phenolics compounds which are antioxidants are responsible for the anti bacterial activity (20). The total phenol contents (TPC) of the methanolic and aqueous extracts of R. damascena flower (Table 1) were comparable to other study (21). The total phenolic contents of fresh (FF) and spent (SF) Rosa damascena flower extracts were reported as 276.02 ± 2.93 and 248.97 ± 2.96 mg GAE/g respectively (3) which is almost double amount of our findings. The higher phenolic acid levels in methanolic extracts could be due to extraction of both nonpolar and semipolar soluble phenolic acids. Many different methods have been established for evaluating the antioxidant capacity of certain biological samples, with such methods being classified, roughly, into one of two categories based upon the nature of the reaction that the method involved (22). The methods involving an electron-transfer reaction include the total phenolics assay using Folin-Ciocalteu reagent, the TEAC and the DPPH radical-scavenging assay. The IC50 value for the methanolic extract of R. damascena was reported relatively as low as 21.4 μg/ml (21). The partially purified acetone fraction of Rosa damascena Mill. flower required for 50% inhibition of superoxide radical production, hydroxyl radical generation and lipid peroxide formation were 13.75, 135 and 410µg/ml, respectively (23). Fresh and spent Rosa damascena flower extracts showed 74.51±1.65 and 75.94±1.72% antiradical activities at 100ppm. (3) which are lower than those of our extracts. DPPH is a stable free radical that can accept an electron or hydrogen radical to become a stable diamagnetic molecule. A significant correlation was shown to exist between the phenolic content and with DPPH scavenging capacity for each spice (24). Thus, owing to high content of polyphenols, rose extracts showed high antioxidant activities. These phenolic antioxidants play important role as bioactive principles in the rose flowers used as traditional medicines (21). LPI activity is mainly attributed to the hydrophobic character of the antioxidant molecules but total phenolics content (TPC) measures both types of antioxidants, hydrophobic and hydrophilic (15). The high antioxidant activity of the R.damascena extracts could be attributed to its high phenolic content. In vivo evaluation of antioxidant effects of ethanol extract of R. damascena petals performed by oral administration at doses of 50, 75, 100 and 200 mg/kg/day in rats for 10 days showed the highest activity with the dose of 200 mg/kg/day (25). This preliminary study indicates the interesting anti oxidative stress activity of R. damascene suggesting its positive applications as a medicinal source for the treatment and prevention of free radicals associated diseases. R.damascena extract with a high phenolic content and good antioxidant activity can be supplemented for nutritional purposes. Oral administration of acetone fraction at 50mg/kg body weight significantly reduced the serum alkaline phosphatase (ALP), glutamine pyruvate transaminase (GPT) and glutamine oxaloacetate transaminase (GOT) activity and lipid peroxide level in rats receiving an acute dose. This indicated that R. damascena could protect against induced hepatotoxicity, possibly by its free radical scavenging activity (23). The extract displayed an excellent cytotoxic action towards the human tumor cell line. Although all in vitro experiments hold limitations with regards to possible in vivo efficacy, the results of this study are very promising with regards to possible anti-neoplastic chemotherapy and form a very sound basis for future research. Some reports support the relationship of cytotoxicity with antioxidant activity (26). So the antioxidant activity of R. damascena extract might contribute to its cytotoxic activity.
Plants contain a wide variety of antioxidant phytochemicals or bioactive molecules, which can neutralize the free radicals and thus retard the progress of many chronic diseases associated with oxidative stress and reactive oxygen species (ROS). The intake of natural antioxidants has been associated with reduced risk of cancer, cardiovascular disease, diabetes and diseases associated with ageing. It can be concluded from the above results that R. damascena extract exhibited antimicrobial activity only against S.aureus. The extracts provided better antioxidative activity as compared with synthetic antioxidants, which provides a way of screening antioxidants for foods, cosmetics and medicine. Hence, the R.damascena extract may be exploited as a natural antioxidant and health promoting agent that can conveniently finds its appropriate therapeutic applications.