Oleo gum resin of C. molmol was obtained from Gattefossé SAS Co., France. The oleo gum resin was grounded into coarse powder by a grinder. Voucher specimen was kept at the Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt (labeled M-12).
Preparation of the hexane extract
Myrrh powder (500 mg) was extracted using hexane by maceration for 24 h at room temperature with occasional shaking (250 g × 2 L). The extracts were then filtered and dried by rotary evaporation.
Extraction of the essential oil
Myrrh powder was subjected to hydro-distillation for 5 h using a Clevenger apparatus. Obtained essential oil was dried over anhydrous sodium sulfate. The oil was refrigerated at 4 °C in a sealed amber vial till use.
Source of microorganisms and culture media
Three standard laboratory reference strains from American Type Collection Culture (ATCC) for bacteria (purchased from IMTECH, Chandigarh, India) were used for determination of the bactericidal activity. The tested microorganisms are listed in Table 2. Cultures were adjusted to 0.5 McFarland standard which contains approximately 1 to 2 × 108 CFU/ml with tested bacterial strains, then dilute the 0.5 McFarland suspension 1:10 in sterile broth or saline to obtain a concentration of 107 CFU/ml, the adjusted suspensions for final inoculation should be used within 15 min of preparation.
Four multi-resistant clinical isolates (Table 3) were obtained from Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Alexandria, and their identity was ascertained by classical procedures (Berkowitz and Jerris 2015).
Determination of bactericidal activity by viable count technique
Stable stock emulsion of the oil was prepared using Cremophor El (triturating ten volumes of the oil with one volume of Cremophor El (10 ml oil + 1 ml Cremophor El) (Wisher 2012). The resultant emulsion was sterilized by filtration though 0.45 µm membranes filter (Millipore, USA). Aliquots of each emulsion were properly diluted with sterile water, inoculated with overnight culture of test organism diluted with water (1:100), vortexed and incubated at 37 °C. The same volume of Cremophor El completed to 100 ml with water was used as a negative control. At specified time intervals, the inoculated systems were vortexed, and aliquots were decimally diluted with sterile saline and the number of viable cells was determined by transferring 20 µl portions of each dilution onto the surface of overdried soybean casein Digest agar (Oxoid) plates (Sigma, USA). These were incubated at 37 °C for 48 h and the number of developed colonies was counted and average number of cells calculated as CFU/ml. Controls lacking tested products were included in the test. For determination of the effect of MEO emulsion concentration, the same procedure was followed and oil concentration varied between 0 and 5% v/v.
Determination of antibiotic resistance
Antibiotic resistance pattern of clinical isolates was determined by single disk agar diffusion technique using 26 different Oxoid-made antibiotic susceptibility disks and Muller Hinton agar (Oxoid). The resultant inhibition zones were translated into antibiotic resistance pattern using published tables.
Preparation of the cream and determination of its bactericidal activity
The cream was made up of the following [g%]: MEO emulsion , stearic acid , stearyl alcohol [1.0], cetyl alcohol [1.0], potassium hydroxide , glycerol  and distilled water [Q.S to 100].
One gram quantities of the prepared cream were distributed into sterile small beakers. Each beaker then received 100 µl of an overnight culture of the organism and mixed thoroughly using sterile glass rods under laminar air flow cabinet. At the specified times, 9 ml of sterile water were added to each inoculated cream, mixed well, decimally diluted with sterile water and the number of viable cells was determined by surface viable count technique. As a control a placebo cream was included in the test.
Preparation of the mouthwash and determination of its bactericidal activity
Mouthwash was made up of the following [g%]: MEO emulsion , cremophor El [2.5], lidocaine HCl [0.01], citric acid [0.03], sodium citrate [0.07], saccharin sodium [0.02] and distilled water [Q.S. to 100 ml].
10 ml of mouthwash were decimally diluted with sterile saline and used for the determination of the number of surviving organisms using the described surface viable count method. The test was repeated using placebo mouthwash as control. The percent recovered of microbial cells (CFU/ml) was plotted against time following application of mouthwash. Listerine® was used as a standard mouthwash for comparison.
In-vitro cytotoxic activity
Human tumor cell lines
Hep G2 (human liver cancer), HCT-116 (human colon carcinoma) cell lines and MCF-7 (human breast adenocarcinoma) cells, maintained in the laboratory of Cancer Biology Department of National Cancer Institute in Egypt, were used for in vitro cytotoxicity assay. A control was included using normal hamster lung fibroblasts (V79 cells).
Different concentrations of the essential oil and hexane extract (0–200 µg/ml) were tested for cytotoxicity against the selected human cancer cell lines using sulforhodamine B stain (SRB) method (Skehan et al. 1990). The relation between survivals and the oil concentration was plotted to get the survival curve of each tumor cell line after the application of specific concentration. The results were compared to those of the standard cytotoxic drug, doxorubicin (10 mg adriamycin hydrochloride, in 5 ml IV injection, Pharmacia, Italy) at the same concentrations were used as standard cytotoxic agent. The dose of the test solutions which reduced survivals to 50% (IC50, µg/ml) was calculated together with selectivity index.
Gas chromatography/mass spectrometry (GC/MS) analysis of the essential oil and hexane extract
An Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) with a capillary column RTX-5MS (30 m × 0.32 mm, film thickness 0.25 μm) was used for the GC/MS analysis of the essential oils. This was coupled to an Agilent 5975C mass selective detector. The initial oven temperature was 40 °C for 2 min, then it was raised at the rate of 5 °C/min until it reached 210°. The injector and detector temperatures were 290 and 300 °C, respectively. Helium carrier gas was used at a flow rate of 2 ml/min. Manual split mode injection was applied (0.1 μl, each). EI mode was used for recording the mass spectra. The range for m/z was 35–500. Ionization voltage was 70 eV and ion source temperature was set at 230 °C. The above conditions were applied for the analysis of a homologous series of n-alkanes to calculate Kovat’s index (KI). Identification was based on comparison of KI with literature (Adams 2007), in addition to obtained data from Wiley’s MS libraries. Authentic compounds (Sigma-Aldrich, Germany) were also used for identification of some compounds.
Gas chromatography/flame ionization detection (GC-FID)
The GC analyses were carried out on a Focus GC® (Thermo fisher scientific®, Milan, Italy) equipped with TR5-MS fused bonded column (30 m × 0.25 mm × 0.25 µm) (Thermo fisher scientific®, Florida, USA) and FID detector; carrier gas was nitrogen (1.5 ml/min); the operating conditions were: initial temperature 40 °C, 1 min isothermal followed by linear temperature increase till 230 °C at a rate of 4 °C/min, then 5 min isothermal. Detector and injector temperatures were 300 and 220 °C, respectively. The split ratio was 1: 20. Chrom-card® chromatography data system ver. 2.3.3 (Thermo Electron Corp. ®, Florida, USA) was used for recording and integrating of the chromatograms. Average areas under the peaks of three independent chromatographic runs were used for calculation the % composition of each component.
Tests were conducted in triplicate and values recorded as mean ± SEM. Results were analyzed by GraphPad Prism® v.5 software. Significant differences among means of different samples were analyzed using paired-t-test at p ≤ 0.05 for all analysis except cytotoxic analysis which was separated using Bonferroni posttests at p ≤ 0.05.