Citrus lemon as pruning materials were collected on February 2017 from a private farm situated in Nubaria district. The pruning wastes composed of leaves were freshly weighed, then oven-dried at 50 °C and again weighed. Water distilled using a Clevenger apparatus was used to collect higher quantity to determine volatile oils percentage, and then GC–MS analyses of the volatile oils were adopted.
GC–MS analyses of the volatile oils obtained from Citrus lemon leaves
The GC–MS system (Agilent Technologies) was equipped with gas chromatograph (7890B) and mass spectrometer detector (5977A) at Central Laboratories Network, NRC Cairo, Egypt. Samples were diluted with hexane (1:19, v/v). The GC was equipped with HP-5MS column (30 m × 0.25 mm internal diameter and 0.25 μm film thickness). Analyses were carried out using helium as the carrier gas at a flow rate of 1 mL/min at a split ratio of 1:10, injection volume of 1 µl and the following temperature program: 40 °C for 1 min; rising at 4 °C/min to 150 °C and held for 6 min; rising at 4 °C/min to 210 °C and held for 1 min. The injector and detector were held at 280 °C and 220 °C, respectively. Mass spectra were obtained by electron ionization (EI) at 70 eV; using a spectral range of m/z 50–550 and solvent delay 5 min. Identification of different constituents was determined by comparing the spectrum fragmentation pattern with those stored in Wiley and NIST Mass Spectral Library data.
Bacterial stains and culture media
Four bacterial strains of importance were used to trial the antibacterial properties of the volatile oil obtained. Two of the bacterial strains were Gram-positive Staphylococcus aureus NRRL B-313 and Bacillus cereus NRC, while the others were Gram-negative Escherichia coli NRC B-3703 and Pseudomonas aeruginosa NRC B-32. Nutrient agar media was used in this study for bacterial strains growth. The media was sterilized at 121 °C by autoclaving after that used for sub-culturing, and agar media was utilized for agar well diffusion assay.
Agar-well diffusion assay
About 20 mL nutrients of agar media were sited into Petri dishes (10 mL). Tween 20 (0.5% v/v) was added to the agar after autoclaving to improve oil solubility, and 100 μL of the fresh cultures was spread over the plate using a sterile swap spreader to get a uniform microbial growth for all plates. A well was done as 9 mm diameter in the agar plate. The wells loaded with 50 μL of the Citrus lemon leaves volatile oil. The plates were left for 1 h at refrigerator to allow the diffusion of oil, and then plates were incubated at 37 °C for 24 h. The inhibition zone appeared was measured with a ruler.
Minimal inhibitory concentration (MIC) determination
Determination of the MIC for Pseudomonas aeruginosa and Staphylococcus aureus was measured by optical density assay for the different microbial strains P. aeruginosa and S. aureus by added different volumes of volatile oil 1, 2, 3, 4, and 5 μL. C. limon leaves volatile oil at 5 mL nutrient broth culture was then inoculated with 50 μL of the S. aureus and P. aeruginosa fresh cultures and incubated in shaking incubator at 150 rpm at 37 °C for 24 h. Microbial growth was measured at 620 nm, and the results were expressed as growth inhibition percentage.
Mode of action
The effects of different concentrations of lemon leaves volatile oil on several biochemical activities were studied according to Ramadan et al. (2012). After inoculating flasks with S. aureus and P. aeruginosa strains, during the middle logarithmic growth phase, volatile oil was applied to lemon leaves at concentrations of 1/8, 1/4, 1/2 MIC. Then, the flasks were shaken at 120 rpm at 37 °C. Samples were withdrawn at the onset of the experiment and after incubation periods of 20, 40, 60, 80, 100, and 120 min in the case of P. aeruginosa, and 20, 40, 60, 80, 100, 120, and 180 min in the case of S. aureus. The bacterial cells were subjected to determination of acid-soluble phosphorus, total lipids, soluble protein, DNA, and RNA.
Acid-soluble phosphorous was determined according to Hogeboom and Schneider (1950) and Toribara et al. (1956). Bacterial cells were collected, washed two times with ice-cold saline and extracted two times with 5% ice-cold trichloroacetic acid. The suspensions were finally centrifuged at 5000 rpm. 1 mL of extract was added to 4 mL reagent (40 mL of 6 N H2SO4, 80 mL distilled water, 40 mL ascorbic acid, and 40 mL from a solution of ammonium molybdate), mixed and incubated for 2 h at 37 °C, and then cooled to room temperature. The absorbance was measured at 680 nm. Total lipid was determined according to Bligh and Dyer (1959) and Kinght et al. (1972). The residue after removing of the acid soluble phosphorous was extracted three times with chloroform: methanol mixture (2:1, v/v). 0.1 mL of volatile oil was added to 5 mL of concentrated H2SO4. The mixture was heated in a water bath for 10 min and then cooled, and 0.4 mL aliquot was placed in a dry test tube. 6 mL of phosphor-vanillin reagent (0.6 gm vanillin dissolved in 10 mL ethanol before diluting to 100 mL with distilled water was mixed with 400 mL of concentrated orthophosphoric acid) was then added to each test tube. The mixture was set in the dark for 45 min, and the absorbance was measured at 525 nm. Soluble protein was determined according to Bradford (1976). The de-lipidated cells were solubilized in 1 N KOH for 20 h at 37 °C. The soluble proteins were determined at 595 nm. RNA was extracted according to Burton (1957) and Malik and Singh (1980). The residue of the sample after hydrolysis by 1 N KOH was subjected to extraction of RNA. To each sample, HCl (6 N) was added, and then the solution was completed with the same volume of 10% TCA. After adjusting the concentration, then the residue was washed with 5% TCA. 1 mL of RNA was added to 3 mL of reagent (0.2 gm orcinol was dissolved in 15 mL distilled water, and 135 mg of ferric ammonium sulfate, then 85 mL of concentrated HCl was added), mixed, and heated in a water bath for 20 min. The tubes were cooled and measured at 670 nm. DNA was extracted according to Burton (1957 and Malik and Singh (1980). Remaining portions after extraction of RNA were hydrolyzed by 5% TCA, and the supernatants were heated for 30 min at 90 °C, cooled, and centrifuged at 5,000 rpm. The residue was washed one time with 5% TCA. 1 mL of DNA extract was added to 2.5 mL of the diphenylamine reagent (1 gm of diphenylamine was dissolved in 98 mL of acetic acid, and then 2 mL of H2SO4 was added), and the mixture was heated for 5 min in a water bath. The samples were cooled, and absorbance was measured at 540 nm.