COMPOSITION AND ANTIMICROBIAL ACTIVITY OF THE ESSENTIAL OIL OF

Ruta chalepensis L. is an aromatic plant belonging to the family Rutaceae, widely distributed in Saudi Arabia. The study focuses on the extraction by steam distillation, phytochemical analysis, GC/MS analysis and antimicrobial activity of essential oil from the leaves of the plant. The results revealed the presence of flavonoids, terpenoids, steroids,and sugars at reasonable concentrations in ethanol, chloroform, ethyl acetate, petroleum ether and n-butanol extract of R. chalepensis leaves. GC/MS analysis identified twenty-eight compounds with the major compounds being 2-Tridecanone (38.88%), 2-Nonanone (29.56%), 2-Acetoxytridecane (18.58%), while 2-Pentadecanol acetate (3.42%), 2-Decanone (1.67%) and 2-Dodecanone (1.25%) were found in small concentrations and the other compounds were found in trace amounts of less than 1% for each. The antimicrobial activity determined by disc diffusion methods showed that the highest activity was observed against S. aureus (inhibition zone of 20.33±0.151 mm), followed by E. coli (inhibition zone of 16.67±0.158 mm), Bacillus cereus and Candida albicans (inhibition zones of 15.67±0.254 mm and 15.33±2.52 mm, respectively) and the lowest activity was against Pseudomonas aeruginosa (inhibition zone of 14.67±1.64 mm). The study, therefore,highlighted on the fact that extracted essential oil could be considered as a potential antimicrobial agent in therapeutic and pharmaceutical applications.

Essential oils are the most important herb and plant compounds which are rich in hydrocarbon compounds with oxygenated, hydrogenated and dehydrogenated functional groups (Jaradat et al., 2017). Most of these chemicals are odorous mono-or sesquiterpenoids, being, found in different parts of the plant and evaporate at normal temperatures (Jaradat et al., 2017). The biological activities of R. chalepensis may be associated with the presence of natural products such as alkaloids, coumarins, phenols, saponins, flavonoids, triterpenes and essential oils (Boudjema et al., 2018). These compounds are complex natural volatile liquids characterized by a strong odour, rarely coloured, soluble in lipid and organic solvents, synthesized by buds, bulbs, leaves, stems, twigs, seeds, fruits, roots, and wood or bark and deposited in secretary cell cavities, canals, epidermal cells or granular trichomes (Boudjema et al., 2018). Numerous studies tested the antimicrobial activity of R. chalepensis essential oil against Gram-negative, . In this study, the antimicrobial activity and total phenolic content of R. chalepensisessential oil wereinvestigated and the chemical composition was determined using (by) GC/MS method.

Materials and Methods:-
Sample Collection: R.chalepensis L. leaves were collected from Al-Baha area, Kingdom of Saudi Arabia and used to prepare the oil extracts. The plant was taxonomically identified and authenticated by Dr. Haidar Abd Algadir, Department of Biology, Faculty of Science, Al-Baha University, where the voucher specimen was deposited for future reference. The leaves were rinsed with freshwater to remove soil and dust particles and sliced into small pieces.

Essential Oil Extraction:
The essential oil was extracted from fresh leaves (300 gm) by hydro-distillation using a Clevenger apparatus at 350°C for 4 hr (Majdoub et al., 2014). The extracted essential oil was dried over anhydrous sodium sulphate (Na 2 SO 4 ) and further conserved at 4 °C in sealed opaque bottles until analyzed (Boudjema et al., 2018).

Preparation of Ethanolic Extract:
The fresh leaves of the plant were cleaned, dried under shade for 20 days and subsequently powdered. The ground material (500 g) was extracted with 80% ethanol and left for three days. The sample was then filtered and the collected filtrate was left to dry at room temperature for 10 days (Amabye and Shalkh, 2015). The extract was stored in a coloured bottle at 4-6°C till analysis.

Fractionation of the Ethanolic Extract by Liquid-Liquid Extraction:
The ethanol extract was dissolved in 200 mL distilled water and further extracted with petroleum ether, chloroform, ethyl acetate, and n-butanol, respectively. After evaporation of the solvents, the solid extracts were collected and used for the purposes of phytochemical analysis and antimicrobial activity test (Amabye and Shalkh, 2015).

Analysis of Essential Oil by GC/MS:
Various essential oil components were identified and quantified by a gas chromatography/mass spectrophotometer (GC/MS-QP2010 ultra, Shimadzu Company, Japan). The sample (0.1 μL) was injected in split mode (a ratio of 1:10) into the capillary column (30 m x 0.25 mm x 0.25 μL film thickness). The instrument was operating in EI mode at 70eV. The procedure was carried out using helium as a carrier gas with a flow rate of 1.69 ml/min, and the oven temperature was programmed from 50°C at a rate of 7°C/min to 180°C and from 10°C/min to 280°C and then 1125 kept for 28 min. The injection port temperature was 300°C. The temperature of the ion origin was 200°C, while the interface temperature was 250°C. An electron ionization device with a detector volt of 1.7 kV was used for GC/MS detection. The sample was analyzed using scan mode in the range of 40-500 m/z. Chemical constituents of essential oils were identified by contrasting their MS with the reference spectra in the mass spectrometry data center of the National Institute of Standards and Technology (NIST) and their retention and Kovats indices were compared with the literature. Quantitative data were collected digitally from area percentages and combined peaks without the use of a correction factor (Boudjema et al., 2018).

Preparation of Bacterial Suspensions:
Aliquots (1 ml) of a 24 hr bacterial broth culture were aseptically distributed onto nutrient agar slopes and incubated at 37ºC for 24 hr. The bacterial growth was harvested and rinsed off with 100 ml sterile normal saline to produce a suspension containing about 10 8 -10 9 cfu/ ml, which was stored in the refrigerator at 4°C till used. The average number of viable organisms per ml of the stock suspension was determined by surface a viable quantification technique (Miles and Misra, 1938). Serial dilutions of the stock suspension were prepared in sterile normal saline solution and 0.02 ml volumes of the appropriate dilution were transferred by micropipette onto the surface of dried nutrient agar plates, which were allowed to stand for 2 hr at room temperature for the drops to evaporate and then incubated at 37°C for 24 hrs. After incubation, the number of developed colonies in each drop was counted, and the average number of colonies per drop (0.02 ml) was multiplied by 50 and the dilution factor to obtain the viable count of the stock suspension, expressed as cfu/ml suspension. Each time a fresh stock suspension was prepared. All the above experimental conditions were constantly maintained constant so that suspensions with very close and accurate viable counts would be obtained.

Preparation of Fungal Suspension:
The culture of C. albican was grown on Sabouraud dextrose agar and incubated at 25°C for 4 days, then harvested, washed with sterile normal saline solution and stored in 100 ml sterile normal saline solution at 4°C till used.

Agar Disc Diffusion Method:
The disc diffusion method was utilized to test the antibacterial activity of the extracts onMueller Hinton agar (MHA) and Sabouraud dextrose agar (SDA) media (Boudjema et al., 2018). Bacterial and fungal suspensions were diluted with sterile physiological solution to 10 8 cfu/ ml (turbidity = McFarland standard 0.5). One hundred microliters (100 µL) of bacterial and fungal suspensions were swabbed uniformly on the surface of MHA and SDA and the inoculum was allowed to dry for 5 minutes. Sterilefilter paper discs (Whatman No.1, 6 mm in diameter) were placed on the surface of MHA and SDA and soaked in 20 µl extracts. The inoculated plates were incubated at 37°C for 24 hr in an inverted position. After the incubation period, the antimicrobial activity was determined as diameter of inhibition zone as follows: <9 mm zone (resistant strain); 9-12 mm (partially sensitive strain);13-18 mm (sensitive strain); >18 mm (very sensitive strain).

Statistical Analysis:
The statistical analyses were performed using Statistical Analysis Systems (SAS, Ver. 9, SAS Institute Inc., Cary, NC, US) and the results were presented as the mean ± standard deviation (SD) of three replicates.All the data were statistically assessed using the General Linear Model (GLM) and the significant difference was performed using Duncan multiple range test at P≤0.05.

Results and Discussion:-Phytochemical Analysis:
The current study showed that R. chalepensis leaves extracted with ethanol, chloroform, ethyl acetate, petroleum ether, and n-butanol contain flavonoids, terpenoids, steroids and sugars at appropriate concentrations with the exception of tannins in petroleum ether extract and saponins in chloroform and petroleum ether extracts. Alkaloids are present in ethanol, chloroform, and petroleum ether extracts, while ethyl acetate and n-butanol extracts were not tested (Table 1)

Chemical Composition:
The essential oil was obtained by steam distillation of fresh leaves, and the chemical composition was analyzed by GC/MS, and the results are presented in Table 2and

Antimicrobial Activity:
The antimicrobial activity of R. chalepensis L. essential oil against Gram-negative (Escherichia coli, Pseudomonas aeruginosa), Gram-positive (Staphylococcus aureus, Bacillus cereus) bacteria and fungi (Candida albicans) is presented in Table 3. The disc diffusion method is based on the measurement of the clear zone surrounding colonies due to inhibition of growth produced by a film disc containing the antimicrobial agent when in direct contact with a bacterial culture. There is a significant variation (P<0.01) in the antimicrobial activity of the essential oil against microorganisms tested, with the highest activity being observed against S. aureus (inhibition zone of 20.  Boudjema et al. (2018) reported that R. chalepensis essential oil reacts differently against all strains studied, and both yeast strains (S. cerevisiae and C. albicans) tended to be more sensitive than others, with inhibition zones ranging from 27 mm to 28 mm. Abdelwahab et al. (2011) reported that the essential oil extracted from R. montana (Clus.) L. prevented the growth of the tested microorganisms with the inhibition zone increasing proportionally with the concentrations of the tested samples. The inhibition zone varied from 9.33 to 24 mm with the highest inhibition activity being recorded againstStaphylococcus aureus at 2 mg/ml, and a considerable inhibition activity with the same concentration againstKlebsiela pneumoniae.Franca Orlanda and Nascimento (2015) reported a wide variation in the antibacterial properties of essential oil against different bacteria,and the highest activity was observed against B. cereus and S. aureus(25.60±0.03 and 22.00±0.06 mm, respectively), moderate activity was against M. flavus, E. coli, M. luteus, E. 1127 aerogenes and S. typhi, and the lowest activity was against P. aeruginosa(8.30±0.05 mm). Jaradat et al. (2017) reported that the essential oil of R. chalepensis from Jerusalem, Hebron, and Jenin regions of Palestine extracted by microwave-ultrasonic method demonstrated activity against the growth of all microbes studied. The highest antibacterial activity (lowest MIC) was for R. chalepensis essential oil from Jerusalem against E. coli, P. aeruginosa and S. aureus, while the highest antifungal activity was for R. chalepensis essential oil from Jenin region against C. albicans (Jaradat et al., 2017).

Conclusion:-
R. chalepensis leaves have been shown to possess reasonable concentrations of flavonoids, terpenoids, steroids, and sugars when extracted with ethanol, chloroform, ethyl acetate, petroleum ether, and n-butanol. The qualitative and quantitative analysis of GC/MS oil extract identified twenty-eight (28) compounds with an abundance of 2-Tridecanone, 2-Nonanone, and 2-Acetoxytridecane. The essential oil had an antimicrobial effect against S. aureus, E. coli, B. cereus, C. albicans, and P. aeruginosa. To the best of my knowledge, this investigation was the first endeavour to studythis species in Al-Baha area, and the findings of both the chemical constituents and the antimicrobial activity varied substantially from those of earlier studies.