Plant material
The A. argutafruits were collected from the experimental farm of the Shenyang Agricultural University in September 2019.
Microbial strains
The AEO were tested against six microorganisms, including two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) as well as two fungal strains (Saccharomyces cerevisiae and Microsporum canis). The susceptibility of the bacteria and the fungi to the essential oils were carried out using the disk diffusion method. All the strains were obtained from the Agricultural Culture Collection of China.
Human cell lines
Human colon cancer cell line (HT-29), human prostatic cancer cell line (PC-3), and human lung adenocarcinoma epithelial cell line (A549) were obtained from the College of Basic Medicine of China Medical University (Shenyang, China). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. The cells were grown at 37 °C in 95% humidified air and 5% CO2.
Extraction of essential oil
The fruits were grinded into a homogenate and subjected to solvent extraction by N-hexane(Ghahramanloo et al. 2017). The obtained AEO was stored at 4°C in sealed glass vials. Percentage AEO yield was quantified as follows:
AEO yield (%) = [mass of AEO obtained (g)/ mass of fresh fruit (g)] ×100
Gas Chromatography-Mass Spectrometry (GC-MS) analysis
The chemical composition of the essential oil was analyzed by GC/MS using Agilent 5973 EI mass selective detector coupled with Agilent GC6890, equipped with a HP-5MS fused capillary column (5% phenyl Methyl Silox) (30m×0.25mm, 0.25μm film thickness). Helium (99.999%) was used a carrier gas with a flow rate of 1.0 mL/min. The initial temperature was programed at 40°C, then increased 3°C/min up to 80°C, then by 5°C/min up to 280°C. The temperature was maintained at 280°C for 20min, just as the injector and detector temperatures. The quadruple mass spectrometer was scanned over a range of 35-500amu at 1 scan per second, with a temperature of 150°C, ionizing voltage of 70 eV, and an ionic source temperature of 230°C. 2.0 μL of the AEO was injected with a split ratio of 10:1. Individual components of the AEO were identified on the basis of their retention indices (RI), and the compared with reference data using the Wiley7n.l library.
Antimicrobial activity assay
Agar diffusion method
The effect of the AEO on the bacteria was determined according to Marjana(Radunz et al. 2019), with few modifications. The bacterial cells were cultured in liquid Luria-Bertani media overnight at 37°C, while the fungal strains were cultured in Sabouraud dextrose broth at 28°C for 48h. The microbial suspensions were diluted to 108 CFU/mL, while the fungal cells were diluted to 106 CFU/mL. The microbial suspension (150 μL) were evenly spread on solid media. Thereafter, sterile 6mm diameter filter disks were placed on the media seeded with the microorganisms (3 disks per plate) and then AEO was dropped onto each paper disk (40 μL per disk). The treated plates were first kept at 4°C for 1h, then incubated at 37°C for 24h (bacteria), or at 28°C for 48h (fungi) (Lu et al. 2007). The antimicrobial activity was evaluated by measuring the diameter of growth inhibition zone surrounding the disks. All tests were performed in triplicates.
MIC and MBC/MFC
The AEO was dissolved in 1% (v/v) DMSO and then diluted to different concentrations (0.78-12.5 mg/mL). Minimum inhibitory concentration (MIC) value was measured following a protocol described by (Zhao et al. 2018), with slight modifications. Briefly, 10 μL from each of the incubated suspensions were transferred into the corresponding media and incubated at 37°C for 24h (bacteria) or at 28°C for 48h (fungi). The minimum concentration that inhibited the growth of the microorganisms was recorded as MBC (minimum bactericidal concentration) or MFC (minimum fungicidal concentration) (Ksouda et al. 2019). The experiments were done in duplicates.
Antioxidant activity
The antioxidant activity of the AEO was tested using the following spectrophotometric methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radical scavenging assays as described by (Lu et al. 2018), as well as the β-carotene bleaching test by Wang et al., 2008, with minor modifications. The AEO samples were tested at concentrations of 12.5 to 800 μg/mL and in triplicates. Butylated hydroxytoluene (BHT) was used as the positive control. IC50 values were defined by linear regression analysis and depicted as means ± SD of the triplicates.
Cytotoxicity assay
Determination of IC50
MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay (Hamdi et al. 2018) was used to evaluate the cytotoxicity of the AEO. Briefly, 1×106cells were seeded in 96 well plates for 24 h. The cells were treated with different concentrations of the AEO samples (1-32 mg/mL) for 48 h. Untreated cells were used as the negative control. Up to 0.5 mg/mL of MTT was added into the cells and incubated for 4 h. Thereafter, the medium was replaced by 100 μL of DMSO to dissolve formazan crystals. Absorbance was detected on a StateFax-3200 microplate reader (AEARNESS, CA, USA) using a wavelength of 570 nm and a reference wavelength of 630 nm. The IC50 was calculated by a liner regression analysis with 95% confidence limits. The inhibition of cell proliferation was approximated using the following formula:
Cell growth inhibition ratio (%) = (1-At / Ac) ×100%
where Atis absorbance of the test sample, Acis the absorbance of negative control.
Confocal laser scanning microscopy (CLSM) assay
A total of 1×103A549 cells were seeded in a 6 well plate for 24 h (37℃, 5% CO2). 16 mg/mL of the AEO samples was added and incubated for 48 h. Untreated cells were used as the negative control. The medium was removed, washed twice in cold PBS, and then the cells were digested with 0.25% typsin. The cells were recovered by centrifugation at 3000×g, washed twice in cold PBS, followed by final centrifugation at 3000×g for 5min. The density of the cells was adjusted to 1×103/mL with cold PBS. 1mL of acridine orange (1 mg/mL) was added into the cells and then incubated at room temperature and in darkness for 5 min. A laser confocal microscope was used to image the DNA morphology of the cells.
Statistical analysis
Each of the experiments was performed in triplicate. The mean value was calculated, and the experimental results were expressed as the mean ± standard deviations (SD). Besides, one-way ANOVA in SPSS Statistics 22.0 software (IBM, USA) was used to analyze the significant differences between the data sets. Significance was set at p<0.05.