Chemical composition, bactericidal kinetics, mechanism of action, and antiinflammatory activity of Isodon melissoides (Benth.) H. Hara essential oil

Present study was aimed to investigate the antibacterial activity, bactericidal mechanism of action, killing kinetics and antiinflammatory activity of Isodon melissoides (Benth.) H. Hara essential oil. The gas chromatography-flame ionisation detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of carvacrol (45.4 %), p -cymene (11.6 %) and thymol (11.3 %) as major constituents of the oil. The oil displayed broad spectrum significant antibacterial activity (MIC: 0.13–8.33 ppm; MBC: 0.13– >33.34 ppm) against test strains. The oil exhibited a time and dose-dependent bactericidal effect. The oil disrupted the cell membrane by changing the cell membrane permeability. The essential oil significantly decreased the overproduction of proinflammatory cytokines in LPS-induced inflammation in HaCaT cells without any cytotoxic effect. I. melissoides essential oil can be a promising alternative antimicrobial agent for the control of methicillin resistant staphylococci and other pathogenic bacteria tested, and also useful for the topical anti-inflammatory properties.


GC and GC-MS analysis
The essential oil analysis was carried out using GC-FID and GC-MS techniques. Quantification of the essential oil constituents was done on Centurion Scientific Gas Chromatograph (model CS-5800), equipped with flame ionisation detector (FID) and BP-5 fused silica capillary column (5% phenyl)polymethylsiloxane stationary phase; 30 m  0.25 mm internal diameter; film thickness 0.25 µm).
Oven temperature was programmed from 60°C to 240°C with increase of 3°C min -1 and final hold time of 10 min. The injector and detector temperatures were 240°C and 250°C, respectively. Nitrogen was used as carrier gas at 1.0 mL min -1 . The injection volume was 0.3 µL (diluted in hexane: 1:1) with a split ratio of 1:60. GC-MS analysis was done using a Clarus 680 GC interfaced with a Clarus SQ 8C mass spectrometer of PerkinElmer fitted with Elite-5 MS fused-silica capillary column (5% phenyl)-polymethylsiloxane stationary phase; 30 m × 0.25 mm internal diameter, film thickness 0.25 µm). The oven temperature program was from 60 to 240°C, at 3°C min -1 , and programmed to 270°C at 5°C min -1 . Injector temperature was 250 °C; transfer line and source temperatures were 220 °C; injection size 0.03 µL neat; split ratio 1:50; carrier gas He at 1.0 mL min -1 ; ionization energy 70 eV; mass scan range 40-450 amu. Identification of the essential oil constituents was achieved on the basis of retention index (RI), MS Library search (NIST and WILEY), and by comparing RI and mass spectral data with the literature (Adams, 2007;Chaturvedi et al., 2018).

Antibacterial activity
The antibacterial activity of I. melissoides essential oil was determined against twelve pathogenic  (13419); Pseudomonas aeruginosa (27853) using disc diffusion and micro dilution broth assays as per the CLSI guidelines (CLSI, 2006;CLSI, 2012;Chaturvedi et al., 2018). 100 µL of inoculum was carefully taken and spread on the Mueller Hinton agar plates to get uniform lawn. A total of 8 µL of the sample was impregnated on the sterile paper disc (Himedia labs, 6 mm diameter) and placed over the seeded plates. Plates were stored in incubator at 37 ℃ for 24 h. Net zone of inhibition was determined by subtracting the diameter of disc. The minimum inhibitory concentration (MIC) of the oil samples were determined by broth dilution assay in 96 well micro titre plates. Oil samples were diluted in the range of 100-0.06 µL mL -1 . Two fold serial dilutions was carried out for determining the MIC, last two columns of well was served as control, i.e. 11 th as positive control for bacterial growth and 12 th for plate sterility. 10 µL of freshly prepared working inoculums from overnight culture was inoculated in each well. Plates were stored at 37 ℃ for 24 hrs.

Streptococcus salivarius
Resazurin dye 0.01% was used as an indicator for the MIC determination. Bactericidal end points were determined by spreading a known volume (10 µL) from each well on MHA plate. Tetracycline and Vancomycin were used as positive control, while DMSO was considered as negative control. The experimental observations were taken in triplicate. The bacterial strains were obtained from the Microbial Type Culture Collection Centre (MTCC), Institute of Microbial Technology (IMT) Chandigarh, India.

Bactericidal kinetics
The kinetics of bacterial killing of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), and Enterococcus faecalis (EF) by the essential oil were determined and compared at 2MIC and 4MIC with those of Log-phase culture (1.0 × 10 7 to 2.0 × 10 7 CFU/mL) (Pearson et al., 1980). Aliquots of sample were removed after 0.5, 1, 2, 3, and 6 h and diluted in sterile normal saline solution before plating on Mueller-Hinton agar plates, CFU were counted after 24 h of incubation at 37°C. The experiment was repeated three times and the results presented here are from one of the three independent experiments.

Leakage of proteins through the membrane
The cell integrity of bacteria was examined by quantifying the release of proteins into the supernatant.
The concentrations of proteins in supernatant were determined by using Bradford method (Bradford, 1976). Logarithmic growth phase was estimated for bacterial cells treated with the essential oil at MIC/2, MIC, and 2×MIC except the control, during an incubation period of 24h at 37°C. Microorganisms were separated by centrifugation (10,000 g) for 5 minutes at 4°C, and the concentrations of cytoplasmic proteins was estimated by measuring the optical density at 595 nm. Results was calculated by the linear equation using Bovine Serum Albumin as standard (y = 0.003x + 0.308; r 2 = 0.998).

Leakage of DNA and RNA (Cytoplasmic constituent) through the membrane
The integrity of cell membrane was monitored by the release of cytoplasmic constituents of the cell absorbing at 260 nm (Chen and Stuart, 2002). The bacteria were incubated in MHB at 37°C for 12h.
Logarithmic growth phase cells of bacteria were treated with the essential oil at MIC/2, MIC, and 2×MIC, except the control. Then the samples were incubated at 37°C for 24h. The samples were then immediately centrifuged (10,000 g) for 5 min at 4°C. To determine the amounts of the DNA and RNA released from the cytoplasm, the supernatant was used to measure the optical density at 260 nm. The results are expressed as the absorbance of the sample (treated)-the absorbance of the control (untreated). Data are expressed as mean ± SEM (n = 3).

HaCaT cells
HaCaT, a human keratinocyte cell line was obtained from the National Centre for Cell Sciences Pune, India. Cells were cultured in DMEM supplemented with 10 % fetal bovine serum with antibiotic antimycotic solution in a CO 2 incubator at 37°C with 5 % CO 2 and 90 % relative humidity. HaCaT cells (0.5 × 10 6 live cells/mL) were treated with I. melissoides essential oil at the concentration of 0.3 % and 1 %, respectively, followed by stimulation with LPS for 6 h, separately. Proinflammatory mediators like tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-β (IL-β) in cell culture supernatant were determined by using human-specific enzyme immunoassay (EIA) kits (BD Biosciences, USA) as per the method described (Singh et al. 2014). The values of TNF-α, and IL-6 were expressed as picograms per millilitre.

Measurement of the cell viability
Effect of essential oil on cell viability was carried out in HaCaT cells using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium (MTT) assay (Sharma et al., 2012). Peritoneal macrophage cells (0.5 X 10 6 cells/well) isolated from mice were suspended in RPMI 1640 medium (Sigma, USA) containing 10 % heat-inactivated fetal bovine serum (Gibco, USA) and incubated in a culture 96 well plate at 37°C in 5 % CO 2 in an incubator and left overnight to attach. Cells treated with 1 % DMSO served as a vehicle control for cell cytotoxicity study. The essential oil was dissolved in DMSO. Cells were treated (0.3 % and 1 % essential oil) and incubated for 24 h at 37°C in 5 % CO 2 . After incubation, cells with treatment, 20 µL aliquots of MTT solution (5 mg/mL in PBS) were added to each well and left for 4 h. Then, the MTT containing medium was carefully removed and the cells were solubilized in DMSO (100 µL) for 10 min. The culture plate was placed on a micro-plate reader (Spectramax; Molecular Devices, USA) and the absorbance was measured at 550 nm. The amount of colour produced is directly proportional to the number of viable cells. Cell cytotoxicity was calculated as the percentage of MTT absorption as follows: Percentage (%) of survival = (mean experimental absorbance/mean control absorbance) × 100). Note-a Identification based on the retention index (RI) and mass spectral data (see experimental section); b retention index, determined on BP-5 gas chromatography column; c retention index from literature.