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.