A comparative study on chemical composition and antimicrobial activity of essential oils from three Phlomis species from Uzbekistan

Essential oils obtained from the aerial parts of Phlomis bucharica, P. salicifolia and P. sewerzowii were determined using GC-FID and GC-MS methods. A total of 76 components were identified in the three species representing 97.12, 88.34, and 96.41% of the whole oil, respectively. High percentages of thymol (20.41%) and camphor (14.46%) exist in P. bucharica oil. Methyl palmitate predominates in P. salicifolia oil representing 51.15% whereas thymol (35.76%) is the major constituent in P. sewerzowii essential oil. GC-MS analyses showed that P. bucharica and P. sewerzowii are more closely related comparable to P. salicifolia. The antimicrobial activity of the essential oils was assessed against different microorganisms using agar-disc diffusion and broth microdilution assay. Among the three tested species, the essential oil of P. salicifolia showed the highest antibacterial activity.

column (Restek; 30 m x 0.25 mm i.d., film thickness 0.25 µm, USA). The oven temperature was programmed isothermal at 50°C for 2 min, then raised from 50 to 300 °C at 7 °C/min, and finally held isothermal at 300°C for 10 min; injector temp., 250°C; detector temp., 300°C; carrier gas, He (1.5 mL/min); with split mode (split ratio, 1:20). The sample 0.1 μL was injected automatically to the chromatograph using AOC-20i auto sampler. GC solution® software ver. 2.4 (Shimadzu Corporation, Kyoto, Japan) was used for recording and integrating the chromatograms. Average areas under the peaks of three independent chromatographic runs of each sample were used for calculating the % composition of each component (total peak area = 100%).

GC-MS analysis
Mass spectrum was recorded using Shimadzu GC-2010 plus gas chromatograph (Shimadzu Corporation, Kyoto, Japan) coupled to a quadrupole mass spectrometer Shimadzu QP-2010 equipped with Rtx-5MS fused bonded column (30 m × 0.25 mm i.d. × 0.25 µm film thickness) (Restek, USA) equipped with a split-splitless injector. The capillary column was directly coupled to a quadrupole mass spectrometer. The initial column temperature was kept at 45ºC for 2 min (isothermal) and programmed to 300ºC at a rate of 5ºC/min, and kept constant at 300ºC for 5 min (isothermal). Detector and injector temperatures were 300 and 250ºC, respectively. Helium carrier gas flow rate was 2 mL/min. Mass spectra were recorded applying the following condition: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200ºC. Diluted samples (0.5%, v/v) were injected with split mode (split ratio 1:15). The sample (1 μL) was injected automatically to the chromatograph using AOC-20i auto sampler. GC solution® software ver. 2.4 (Shimadzu Corporation, Kyoto, Japan) was used for recording and integrating the chromatograms. The compounds were identified by comparison of their mass-spectral data and retention indices (RIs) with those of the Wiley Registry of Mass Spectral Data (9 th Ed.), NIST Mass Spectral Library (2011), references (Adams 2007) and with our own Laboratory database (Labib et al. 2017;Mamadalieva et al. 2017;Mamadalieva et al. 2018 ). In addition, the major identified compounds in the essential oil samples were further confirmed by co-injection of some of the available authentic references. The Retention Index (RI) was calculated by relating the retention time of the tested compound "t" to the retention time of coinjected series of straight chain aliphatic hydrocarbons (C 8 -C 28 ) using the following equation:

RI =
Where; RI: retention index. N, N+1: Number of carbon atoms in the smaller and larger n-paraffins bracketing the substance. Rt: adjusted retention time of either the substance or the hydrocarbons

Microbial strains
The antimicrobial activity was evaluated using standard microbial strains: Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (RKMUz 5); Gram-negative bacteria Pseudomonas aeruginosa (ATCC 27879) and Escherichia coli (RKMUz 221); and the fungus Candida albicans (RKMUz 247). The RKMUz microorganism cultures were obtained from the strain collection of the Institute of Microbiology, Academy of Sciences Uzbekistan.

Evaluation of the antimicrobial activity using the disc diffusion method
The antibacterial activity of the essential oils was determined using the modified agar-disc diffusion method. Sterile nutrient agar (25 g agar/L distilled water) was inoculated with bacterial cells (200 µL of bacterial cell in 2 mL 0.9% NaCl suspension and 20 mL medium) and poured into Petri dishes to give a solid medium. Candida albicans (1×10 6 colony forming units per mL) was inoculated into sterile Mueller-Hinton-agar. Forty microliters of test material (5 mg/mL of the essential oils) dissolved in the same solvent used for extraction, was applied on sterile paper discs (Whatman No.1, 6 mm diameter). Ampicillin, ceftriaxone and fluconazole (Himedia Laboratories Pvt. Limited) were used as positive controls and the solvents as negative controls. The solvents were allowed to evaporate in a stream of air. The discs were deposited on the surface of inoculated agar plates. Plates were kept for 3 h in refrigerator to enable the diffusion of the substances into the agar. Plates with bacteria were incubated for 24 h at 37°C and plates with yeasts for 48 h at 26°C. The inhibition zone (including the disc diameter) was measured and recorded after the incubation time. An average zone of inhibition was calculated for the three replicates in independent assays (Wayne 2009a).

Determination of minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MBC)
The broth micro-dilution method was used to determine MIC and MBC as described by Clinical and Laboratory Standards Institute (CLSI) (Wayne 2009b). Firstly, 5% (v/v) dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) as an emulsifier and 0.05% (w/v) agar-agar (Merck, Darmstadt, Germany) as a stabilizer of the essential oil were added in 10 mL Mueller-Hinton Broth (MHB, Merck, Darmstadt, Germany) medium. After autoclaving of medium, different concentrations of the essential oil (0. 017, 0.035, 0.07, 0.156, 0.312, 0.625, 1.25, 5, 10, 20, and 40 g/mL) were set up in Mueller-Hinton broth (MHB) medium. The 96-well plastic microdilution tray with round bottom wells were prepared by dispensing into each well 90 L of MHB containing different concentrations of the essential oil and 10 L of the bacterial inoculum, which was approximately 1×10 6 CFU/mL. Wells containing DMSO and inoculums were used as negative control, whereas ampicillin, ceftriaxone and nystatin consist of positive control. The lowest concentration of the essential oil showing visually no growth (by comparing with the first growth control) was taken as MIC. Determination of the minimum concentration of the essential oil that reduces 99.99% of population bacteria (MBC) was done by culturing of 10 L of each well without any invisible growth. The culturing was performed on Mueller-Hinton agar medium and incubated at 37°C for 24 h.