Antimicrobial activity of Rosa damascena petals extracts and chemical composition by gas chromatography-mass spectrometry (GC/MS) analysis

Antimicrobial activity of alcoholic and aqueous extracts from Rosa damascena was evaluated against 10 pathogenic microorganisms. Minimum inhibition concentration (MIC), Minimum bactericidal concentration (MBC) and the diameter of inhibition zone (DIZ) were determined by in vitro bioassays using hole-plate diffusion method and broth micro-dilution method (BMD) against Staphylococcus aureus ATCC 25923, Staphylococcus aureus, Pseudomonas aeruginosa ATCC 27853, Psuedomonas aeruginosa, Escherichia coli ATCC 25922, E. coli, Streptococcus pneumoniae ATCC 55143, Acinetobacter calcaoceuticus, Salmonella enteritidis and Aspergillus niger ATCC 16404. While hexane extracts showed very low activity against the test microorganisms, ethanol, methanol and water extracts significantly exhibited antimicrobial activity and inhibited the growth of Gram-positive and Gram-negative bacteria as well as A. niger at all tested concentrations. The most active antimicrobial effect was recorded for ethanol extract of R. damascena against P. aeruginosa ATCC 27853 at MIC and MBC of 62.5 μg/ml (DIZ = 34 mm), E. coli ATCC25922 at MIC and MBC of 62.5 μg/ml (DIZ = 30 mm). MIC and MBC data obtained from the antimicrobial studies were analyzed for significant difference at p<0.05 using one way analysis of variance (ANOVA). The extracted oil of Damascus rose petals were characterized by GC/MS; analysis reported that 30 compounds were present. The predominant components were citronellol (14.829.0%), geraniol (11.3-16.2%) and nerol (11.6%) while the phenyl ethyl alcohol was 1.2%. This study sheds the light on the efficacy of plant extracts to combat pathogens which will help as natural antimicrobial agents.


INTRODUCTION
Infectious disease account for approximately one half of all deaths in tropical countries (Iwu et al., 1999) and they are considered a major threat to human health because of the unavailability of vaccines or limited chemotherapy.Ranked 5th in 1981, infectious diseases continue to be a growing public health concern (Kone et al., 2006) and they became the 3rd leading cause of death in 1992, with an increase of 58% (Pinner et al., 1996).Most of the current antibiotics have considerable limitations in terms of antimicrobial spectrum, side effects and their widespread overuse has led to increasing clinical resistance of previously sensitive microorganisms and to the occurrence of uncommon infections (Cos et al., 2006).Plants used for traditional medicines contain a wide range of substances that can be used to treat chronic as well as acute infectious diseases (Diallo et al., 1999;Ofokansi et al., 2011), and have therefore become sources of important drugs and the pharmaceutical industries have come to consider traditional medicine as a source of bioactive agents that can be used in the preparation of synthetic medicines.
Rosa damascena mill L., commonly known as Damask rose (Kaul et al., 2000), is one of the most important species of Rosaceae family.Rosaceae are well known ornamental plants and have been referred to as the king of flowers (Cai et al., 2005;Nikbakht et al., 2005).At present time, over 200 rose species and more than 18000 cultivars form of the plant has been identified (Gudin, 2000).There are evidences that Rosaceae family is an ancient plant (Nikbakht and Kafi, 2008;Yassa et al., 2009).Some fossils of rose are found in America that is 30 million years old (Yassa et al., 2009).The origin of Damask rose is the Middle East, but the origin of its fragrant oil and extracts is Greece (Arezoomandan et al., 2011).The highest quality rose water is produced in Taif Governorate, KSA.Kaaba (God House) in Mecca, Saudi Arabia, is washed yearly by unique and special rose water of Taif.Rose water is also of high value in the food industry and some special foods are prepared using this product (Nikbakht and Kafi, 2008).The R. damascena has also been used for medicinal purposes (Hongratanaworakit, 2009).Various products and isolated constituents from flowers, petals and hips (seedpot) have been studied in a variety of in vivo and in vitro studies.It has been shown that R. damascena has wide spectrum antimicrobial activities.Essential oil, absolute and hydrosol are important products that showed these effects.Ulusoy et al. (2009) and Boskabady et al. (2011) showed that essential oil and absolute have strong antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, B. subtilis, Staphylococcus aureus, Chromobacterium violaceum and Erwinia carotovora strains.
However, there is no detailed study concerning the antimicrobial activity of R. damascena performed so far.Most of the studies related to the damask rose have focused on the growing techniques, harvest time, and physical and chemical properties of oil.Therefore, the purpose of this study was to determine the in vitro antimicrobial activity against gram negative and gram positive bacteria as well as the chemical compositions of R. damascena volatile oil by using GC/MS to determine the major constituents of high quality rose oil.

Preparation of extracts
The present study was conducted to determine the chemical composition (constituents) and antimicrobial activities of R. Damascena flower extracts.The flowers were collected in April, 2013 during the rose harvest season, from different local farms (AL-Hada and Al-Shefa), Taif governorate-Saudi Arabia.The green parts of roses were removed and the remains were cut into small pieces.Following the collection, part of the flowers was immediately frozen in closed containers at -80°C for further analysis.The rest of the flowers, 0.5 kg of fresh flowers from each accession were used immediately for alcoholic and water extraction.Fresh and dried parts of R. damascena (petals, flowers and seed pods) were either powdered or soaked freshly in five volumes of 50% ethanol, 80% methanol, and 100% hexane by stirring overnight, filtered through Whatman No. 1 filter paper after 72 hand centrifuged at room temperature at 5000 x g.The supernatants were evaporated to dryness at 45‫؛‬C under reduced pressure.Powdered R. damascena (500 g each) were extracted twice overnight with 2500 ml of distilled water at room temperature.The supernatant were collected and evaporated to dryness at 45‫؛‬C under reduced pressure.The yield of each extract was adjusted to be less than 10% (20 g of each alcoholic extract was dissolved in 100 ml distilled water containing 30% DMSO to get concentration 500 µg/ml) and stored in dark sterile bottles and became ready until use.The reconstituted alcoholic extracts were sterilized by filtering through 0.45 μm membrane filter and tested for sterility after membrane filtration by introducing 2 ml of the extract into 10 ml of sterile nutrient broth and incubated at 37°C for 24 h.A sterile extract was indicated by the absence of turbidity in the broth after the incubation period (Ronald, 1995).

Antimicrobial activity
All plant extracts were dissolved in sterile distilled water containing 30% DMSO to a final concentration of 500 µg/ml and sterilized by filtration by 0.45 µm Millipore filters.Antimicrobial tests were carried out in this report by agar well diffusion assays according to the NCCLS citeria.The antibacterial activity was carried out by employing 24 h cultures of E. coli (ur), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), P. aeruginosa (ETT), S. aureus (ATCC 25923), S. aureus (Nasal), S. pneumoniae (ATCC55143), S. enteritidis (ATCC 13076), A. calcaoceuticus (ur) and A. niger (ATCC 16404).Activity of aqueous and alcoholic extracts of R. damascena (RD) was tested separately using Agar well diffusion method (Perez et al., 1990;Murray et al., 1995;Olurinola, 1996;Srinivasan et al., 2001;Dulger and Gonuz, 2004).The medium was sterilized by autoclaving at 120C (15 lb/in 2 ).About 30 ml of the agar medium with the respective strains of bacteria was transferred aseptically into each sterilized Petri plate.The plates were left at room temperature for solidification.A well of 6 mm diameter was made using a sterile cork borer.Antibacterial assay plates were incubated at 37±2C for 24 h.The diameter of the zone of inhibition was measured.Sterile ultrapure water and DMSO were used as negative control.Samples were tested in triplicate and results are expressed as mean ± standard deviation.

Determination of antifungal activity
The agar well diffusion method (Anesini and Perez, 1993) was modified.Potato dextrose agar (PDA) was used for fungal cultures (Figure 1).The culture medium was inoculated with the fungal strain A. niger ATCC 16404 separately suspended in potato dextrose broth.A total of 6 mm diameter wells were punched into the agar and filled with plant extracts and solvent blanks (ethanol, methanol and hexane).Standard antibiotic (Nystatin, concentration 1 mg/ml) was used as positive control and fungal plates were incubated at 28C for 48 h.The diameters of zone of inhibition observed were measured.Antifungal activities were determined by measuring diameter of inhibition zone (DIZ) in mm.Each experiment was repeated thrice and the average values of antimicrobial activity were calculated (Shahidi, 2004).

Broth micro-dilution method (BMD)
MICs and MBCs of R. damascena extracts were determined by using BMD method as described by the NCCLS in flat-bottomed 96well clear plastic tissue-cultured plates (NCCLS, 2003a

Isolation of volatile oil
One hundred grams of dried flowers of each plant were ground in a blender and then subjected to hydrodistillation for 3 h according to the standard procedure described in the European Pharmacopoeia (2004).The oil was solubilized in n-hexane, dried over anhydrous sodium sulphate and stored at +4°C in the dark until tested and analyzed.

Gas chromatography-mass spectrometry (GC/MS) analyses
The essential oils were analyzed on a gas chromatograph (GC)mass spectrometer (MS); Clarus 500 GC/MS (PerkinElmer, Shelton, CT) was used.The software controller/integrator was TurboMass, version 4.5.0.007 (PerkinElmer).An Elite-1 GC capillary column (30 m, 0.25-mmID, 0.25 DF, PerkinElmer) was used.The carrier gas was helium (purity 99.9999%) at a flow rate of 0.9 mL/min (initial 7.6 p.s.i.flow initial 36.2cm/s, split; 50:1).Temperature conditions were: inlet line temperature, 270°C; source temperature, 210°C; trap emission, 100°C; and electron energy, 70 eV.The column temperature program was: 80°C hold for 5 min, increased to 150°C (rate, 5°C/min), and held for 5 min, increased to 270°C (rate, 20°C/min) and hold for 5 min.The injector temperature was 220°C.MS scan was from 45 to 350 m/z.A sample volume of 50 µL was diluted with 500 µL of chloroform, vortexed for 2 s, and a volume of 1 µL was injected for GC-MS analysis.A volume of 50 µL from this diluted sample was trans-ferred to total recovery vial 1-mL, mixed with 50 µL of MSTFA, capped, vortexed for 2 s, heated at 80°C for 30 min using heating block for half insertion of vial, cooled, and a volume of 1 µL was injected for GC-MS analysis.
The total ion chromatogram (TIC) was recorded from 45 -350 m/z.The targeted peaks were extracted by the knowledge of major m/z fragments, averaged masses at peak top, and searched for matched compounds using mass spectrometry data bank NIST2008 database.The percentage composition of the essential oil was computed by the normalization method from the GC peak areas measurements.

Statistical analysis
Comparison of data was performed using the one way ANOVA and is presented as mean ± standard deviation.Comparison of MIC and MBC values, tests were made in triplicate for quantification.Values of p<0.05 were considered significant.

Antimicrobial activities of plant extracts
The results show that Escherichia coli ATCC 25922 was sensitive to all antibiotic disks tested but resistant only to AMC, P. aeruginosa ATCC 27853 was resistant to all antibiotic tested but sensitive only to CEZ.The rest of tested bacterial isolates were sensitive to all antibiotic disks tested.The antibacterial activity of aqueous and alcoholic extracts of flowers petals of R. damascena were observed using agar well diffusion method by measuring the diameter of the growth inhibition zone (Table 1).
The ethanolic extracts of R. damascena (RDEE) showed a positive significant antibacterial activity against all tested bacteria; diameter of zone of inhibition ranged between 15 mm for A. niger ATCC 16404 to 34 mm for P. aeruginosa (ATCC 27853).Methanolic extracts of R. damascena (RDME) showed moderate degree of activity against most bacterial organisms tested (diameter of zone of inhibition ranged) between 13 mm for A. niger ATCC 16404 to 25 mm for Psuedomonas aeruginosa (ATCC 27853) and Salmonella enteritidis (ATCC 13076), while the hexane extracts (RDHE) showed very less effect against tested bacteria.The ethanolic extracts of RDEE showed a highly significant antibacterial activity against P. aeruginosa (ATCC 27853), and E. coli (ATCC 25922).
Hexane extracts of R. damascena (RDHE) showed the lowest degree of inhibition against most of the tested bacteria; diameter of zone of inhibition ranged between 9 mm for A.niger ATCC 16404 to 13 mm for Psuedomonas aeruginosa (ATCC 27853).The antifungal activity was determined by measuring the diameter of zone of inhibition recorded.RDEE, RDWE and RDME were found to have maximum antibacterial and antifungal activity in comparison to hexane extracts.RDEE possessed potent antifungal activity against A. niger showing diameter of zone of inhibition ~ 15 mm while methanolic extracts and Water extracts showed diameter Water extracts of R. damascena showed also a highly significant antibacterial activity against tested organisms.The diameters of zone of inhibition ranged from to 13 to 28 mm.The results of minimum inhibitory concentrations (MICs) revealed that the R. damascena alcoholic extracts exhibited the best anti-bacterial activities towards Gramnegative and Gram-positive bacteria with MIC and MBC values between 62.5 to 250 μg/ml (Table 1).
The minimum fungicidal concentration (MFC) of R. damascena (RD) extracts in different solvents were evaluated against A. niger ATCC 16404.Ethanolic, methanolic and water extracts were found to have maximum antifungal activity in comparison to hexane extracts.Ethanolic extracts of RD (RDEE) showed maximum potency against Aspergillus niger ATCC 16404 at highest MFC value of 62.5 µg/ml while the MFC value of ethanolic and methanolic extracts were 125 µg/ml respectively.Thus, the MBC and MFC of R. damascena (RD) extracts showed a bactericidal and fungicidal behavior of these extracts.The anti-bacterial activity of R. damascena alcoholic and water extracts were higher than that of hexane extract.
In the current study, 0.2 ml of rose oil was obtained from 250 g of dried Taif rose petals processed through steam distillation and the oil obtained was observed to be pale yellow in color, and the yield of essential oil was 0.15% (v/w).

DISCUSSION
Generally, the degree of the plant extract activity is revealed by the size of inhibition zone that is expressed by the diameter of the referred inhibition zone.Due to the simple nature of this assay, this technique is less suitable for more precise quantification purposes.MIC and MBC values were calculated to determine the susceptibility of a range of microbial species to a particular extract (Faleiro, 2011).The Gram-negative Pseudomonas aeruginosa (ATCC 27853), E. coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were the most sensitive strains (MIC of 62.5 µg/ml) for RDEE.Similarly, Staphylococcus aureus (ATCC 25923) was the most sensitive strain to RDME (MIC of 62.5 µg/ml).These results agreed with that obtained by Talib and Mahasneh (2010) who reported that butanol extract of R. damascena showed the highest activity (100% inhibition) against S. typhimurium and B. cereus (MIC 0f 62.5 and 250 µg/ml) respectively, while the aqueous extract of R. damascena was also active against C. albicans (MIC of 125 µg/ml).Also, in a study reported by Ulusoy et al. (2009) in Turkey, it was found that Rose absolute and essential oil had a strong antibacterial activity against E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), B. subtilis (ATCC 6633), and S. aureus (ATCC 6538).Shohayeb et al. ( 2014) recorded a similar results that the essential oil and different extracts of Taify R. damascen possess brood spectram antimicrobial activity against gram positive and gram negative bacteria , acid fast bacteria and fungi with MIC and MBC range from 0.125 to 2 and 0.4 to 0.5 mg/ml, respectively .
The molecular mechanism of action of the R. damascene extracts on Gram-negative bacteria is unknown, but the ethanol extracts can probably inhibit the generation of adenosine triphosphate from dextrose and disrupt the cell membrane (Gill and Holley, 2006).
The high amount of hydrocarbons and the concurrent presence of monoterpenes (β-Linalool) could also contribute to inhibit the microbial DNA gyrase as reported by Cushnie and Lamb (2005).
It is well known that R. damascene components show also better antimicrobial effectiveness against Grampositive bacteria.Due to the composition of outer membrane, plant extracts can alter not only such structures but penetrate within the cell, leading to those alterations, such as the denaturation of proteins and enzymes, the "unbalance" of the K+ and H+ ion concentration, until the modification of the entire cell morphology, which can lead to the death of the microorganism (Marrufo et al., 2013).
Phenolic compounds show generally a good antimicrobial effectiveness against Gram-positive bacteria; their effect is dependent on their amount: at low concentrations they are able to interfere with enzymes involved in the production of energy and at higher concentrations, they can induce the denaturation of proteins (Tiwari et al., 2009) until an irreversible modification of the cell and death.
The comparison of the results with the literature showed significant differences for oils, which can be attributed to ecological factors, genetic differences or the development stages of the plant parts analyzed.

Figure 1 .
Figure 1.Agar well diffusion assay showing the antibacterial activity of different extracts of RD.Each well containing 100 µg of the each extract.

Figure 2 .
Figure 2. GC/MS chromatogram of Rosa damascena essential oil collected from Taif province.
).The MIC was assayed using two-fold BMD method in MH Broth in 96-well plates.Plates contained two fold dilutions of antimicrobial agents at the concentration ranges: 0.5,1, 2, 4, 8, 16, 32, 64, 128, 256, and 512 μg/mL.1% DMSO was used as a solvent control.Plant extracts were diluted to twice the desired initial test concentration (1 mg) with Muller Hinton broth (MHB) (Oxoid, UK).All wells, except the first were filled with MHB (50 μL).Plant extract (100 μL) was added to the first well and serial two-fold dilutions were made down to the desired minimum concentration (32 μg).An over-night culture of bacteria suspended in MHB was adjusted to turbidity equal to 0.5 McFarland standards.The plates were inoculated with bacterial suspension (50 μL/ well) and incubated at 37°C for 24 h.Then the turbidity was observed visually by eyes.MICs and MBCs were determined for ethanol, methanol, hexane and aqueous extracts of R. damascena.MIC was determined as the lowest concentration of plant extract that inhibit the growth of each microorganism.MBC was determined as the lowest concentration of plant extract that prevent the growth of bacteria after sub-culturing on MH agar plates.To determine the MBCs, the suspensions (20 μl) were taken from each well without visible growth and inoculated in MH agar for 24 h at 37C.To determine the Minimal Fungicidal Concentration (MFC), 100 μL of each dilution showing no growth was spread on PDA.The inoculated Petri dishes were incubated at 37C for 48 h for fungal cultures.Tests were performed in triplicate for each test concentration.

Table 1 .
MICs, MBCs and MFCs in μg/mL of RD extracts against nine bacteria and one fungal isolate.
of zone of inhibition ~13 mm.Hexane extracts R. damascena showed very low antifungal activity against Aspergillus niger ATTC 16404 (diameter of zone of inhibition of 9 mm).

Table 2 .
Compounds determined in the oil of Rosa damascena extracted by hydro-distillation.
*Dominant compounds are indicated in bold