Materials and methods
Materials and reagents
Supercritical CO2-extracted MRSO was obtained from our laboratory. Hexane and methanol were chromatographic grade obtained from Tedia (USA). HepG-2 and HT-29 colon cancer cell lines were purchased from Boster (Wuhan, China). All components of the cell culture media were purchased from Thermo Scientific (Massachusetts, USA), except biological grade dimethyl sulfoxide from Sigma (St Louis, USA).
Oil samples
Biqi myrica rubra and red pulp hylocereus undatus were purchased from Xianju and Fujian, China, respectively. After removing the defectives, seeds were separated from the flesh and pressed to remove the juice. Then seeds were washed with water and dried at 40 ℃ to reach constant moisture content. Torreya grandis V. merrilli , camellia and Carya nutt were purchased from Fujian and Anhui, China, respectively. Seeds were dried at 40 ℃ after peeling off the coats to reach constant moisture content. Myrica rubra seed oils (MRSO), torreya grandis seed oil (TGSO),hylocereus undatus seed oil (HUSO), camellia seed oil (CSO), carya nutt seed oil (CNSO) were extracted by supercritical carbon dioxide extraction in our laboratory. The supercritical CO2 extraction in bench-scale was used to extract 10 g of each of the ground seeds at a pressure of 360 bar, temperature of 40 ℃, static and dynamic extraction time of 30 min and 2 hours [14, 15]. Extra virgin Olive oil (EVOO) was purchased from a local grocery, originating from Hojiblanca olive in Andalucía, Spain. And the trees were with 20 years old of age. All samples were stored at -20 ℃ in screw-cap amber bottles and thawed prior to further analysis.
Electronic nose experiment
The portable electronic nose PEN3 (Win Muster, Airsense, Germany) with a detector array of 10 metal oxide semiconductor (MOS) type chemical sensors was used. The electronic nose consists of three parts, sampling vessel, sensors array and data acquisition system (Win Muster v.3.0). The MOS is composed of MOS1 (aromatic), MOS2 (broadrange), MOS3 (aromatic), MOS4 (hydrogen), MOS5 (arom-aliph), MOS6 (broad-methane), MOS7 (sulphur-organic), MOS8 (broad-alcohol), MOS9 (sulph-chlor) and MOS10 (methane-aliph). The results were reported by the data of the responses of ten metal oxide semiconductors, and then analyzed through principal component analysis (PCA), a procedure permitting to project the data in a reduced hyperspace determined by primary components.
Electronic nose analysis was carried out according to the method of Buratti et al. with slight modification [2, 16]. Briefly, samples, each of 0.5 mL, were placed in 15 mL glass jars and then incubated at 40 ℃ for 10 min prior to injection. After the headspace equilibration, the running time was 500 s with the program set to include 20 s of referencing, 300 s of sampling, 60 s of washing, followed by 120 s of referencing. Referencing is a procedure zeroing the background noise of the sensors to correct the baseline. Each sample was analyzed in triplicate.
Chemical analysis
The chemical analyses, namely indices like acid value (AV), peroxide value (PV), iodine value (IV) and saponification value (SV), were carried out according to the international standard ISO 660:2009, ISO 27107:2008, AOCS official methods (Cd 1b-87) and ISO 3657:2002, respectively. All chemicals and solvents used were analytical grade. And AVs, PVs, IVs and SVs of every kind of oils were analyzed in triplicate.
Fatty acid composition of MRSO
Fatty acid profiles were determined by preparation of fatty acid methyl esters (FAMEs). Briefly, 50 mg of the seed oil was reacted with 10% H2SO4-MeOH at 70 ℃, before n-hexane being added, and then FAMEs were extracted with n-hexane by adding water to stop the transesterification. The FAMEs were analyzed with an Agilent 7890A gas chromatograph-mass spectrometer-computer (GC-MS) equipped with a flame ionization detector (FID), controlled by TotalChrom Workstation version. A silica capillary column DB-WAX (30m, i.d. 0.25mm, df, 0.25 µm) was used with a temperature program started at 150℃, increased to 200℃ at a rate of 20℃/min and held at 200℃ for 5 min, and then increased to 240℃ at a rate of 5℃/min and held at 240℃ for 10 min. The injector temperature was 250℃, that of the detector 260℃. Electron ionization (EI) spectra were obtained at 70 eV at 210℃. The injection volume was 1 µL, with a split ratio of 1:10 [17]. FAMEs were identified by comparing their retention times with those of mass spectral library from Wiley. Area under each fatty acid peak relative to the total area of all fatty acid peaks was used to quantify the fatty acids identified. Results are reported as g fatty acid/100g of total fatty acids. All samples were analyzed in triplicate.
Oxidative stability of MRSO
The purposes of the presented investigation being identifying the oxidative stability of MRSO were determined as the functions of the (1) storage temperature, (2) different illumination, (3) antioxidants, and (4) metallic ion. Briefly, using a high throughput laboratory assay [18] added a certain quality of MRSO into sealed reagent bottles. Respectively, every other 24 hours, the PV of MRSO treated in the temperature of thermostat box 25, 40, 50, 60 ℃ was detected in accordance to the IUPAC method 2.102. Added a certain quantity of MRSO into sealed reagent bottles and divided into 3 groups to investigate the effects of ultraviolet sunlight, sunshine and dark at room temperature on the oxidative stability of MRSO, according to the PV of samples determined every other 24 hours. A certain quantity of MRSO was added into glass tubes and separately treated under exposure and sealed conditions in 60 ℃. Every other 24 hours, the PV of MRSO was detected to study the effect of air conditions on its oxidative stability. Certain qualities of MRSO were put into the number of copies. 0.02% of TBHQ, 0.02% of BHA, 0.02% of BHT and 0.02% of PG were respectively added into the copies, which then were placed in 60℃. Every other 24 hours the PV was detected to study the effect of antioxidants on the oxidative stability of MRSO. All samples were analyzed in triplicate.
Antiproliferation properties on human colon cancer cells
Human colorectal adenocarcinoma cells proliferation inhibitions were investigated on the basis of the method descripted by Wang et al. and Xie et al. [19, 20]. The cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 unit/mL of penicillin G and 100 µg/mL of streptomycin sulfate) and cultured at 37 ℃ in 5% CO2.
Cells (10,000 cells/ well) were plated in 96-well culture plates, in total volume of 100µL culture media. After incubation of 24 h, the medium was replaced with 100 µL of the appropriate treatment medium (1, 3, 5, 7 mg of seed oil equivalents/ml). Treatment media were prepared by dissolving MRSO in DMSO to make a concentration of 2 g oil equivalent/ml of DMSO. The seed oils in DMSO were then mixed with culture media to achieve the concentrations of 1, 3, 5, 7 mg of MRSO equivalents/ml of treatment media. Media, for both treatment levels and the control, had a final concentration of 0.2% DMSO, and were filtered through a 0.22 µm pore retrograde cellulose filter prior to treatment of cells [21]. Cells proliferation was studied via 3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) [22, 23]. The absorption values of treatment and control media at 490 nm were taken at 24, 48, 72 and 96 h after initial treatment.