2.4 Supercritical CO2 extraction of Qingjinju
seeds
Qingjinju seeds were first ground (model YC-04B) for 30 s. Approximately
1000 g of the ground seeds were transferred into a supercritical
CO2 extraction apparatus equipped with a 5 L extraction
kettle (HA220-50-06, Nantong Huaan Supercritical Extraction Equipment
Co., Ltd., Nantong, China). The instrument was operated using the
following parameters: extraction temperature, 40 °C; extraction
pressure, 35 MPa; analytical
pressure, 12 MPa; analytical temperature,
55 °C; CO2 flow
rate, 15 L/h; extraction time, 3 h. The seed oils were stored at 3 °C in
a refrigerator.
2.5Physicochemical analysis
of Qingjinju seed oils
The specific gravity, refractive
indices, and color values of the oil samples were measured
in
accordance with the People’s Republic of China (PRC) National Standards
GB 5526-198522,
GB/T5527-201023, and
GB/T22460-200824,
respectively. The acid values, peroxide value, and iodine number were
determined using the PRC National
Standards GB/T
5530-200525, GB/T
5538-200526, and
GB/T5532-200827,
respectively. The saponification numbers and unsaponifiable matters were
evaluated using the PRC National Standards
GB/T5534-200828 and
GB/T5535.1-200829,
respectively.
2.6 Fatty acid
composition of Qingjinju seed oils
The oil samples were first converted to fatty acid methyl esters (FAMEs)
using the method described by Carreau and
Dubacq30.
The FAMEs were then quantified by
gas chromatography-mass spectrometry (GC-MS) using a Trace1310 ISQ
(Thermo Scientific, Waltham, MA, USA) equipped with a flame ionization
detector (FID) and TG-5MS capillary column (30m × 0.25mm i.d., 0.25μm
film thickness). The GC temperature program was as follows: initial
temperature, 80 °C for 1 min; increased to 200 °C at 10 °C/min;
increased to 250 °C at 5 °C /min; increased to 270 °C at 2 °C/min and
held for 3 min. The analytical conditions were: detector temperature,
290 °C; injection volume, 1 μL; flow rate of carrier gas, 1.2 mL/min;
ionization voltage, 70eV; scanning
range, 30-400 amu. To identify and quantify the
FAMEs, FAME mixture standards
(35 components, C8-C24) were
injected into the instrument under the same conditions. The FAMEs were
considered positively identified if their mass spectra and retention
indices were comparable to the FAME mixture standards. Quantification of
the samples was performed using the data handling software of the GC/MS
system.
2.7 Volatile
compounds of Qingjinju seed oils
A measured quantity of oil samples were transferred to 10 mL vials
containing 3 mL saturated sodium chloride solution and balanced at 37 °C
for 30 min. (SPME) was performed using a 100 μL polydimethylsiloxane
(PDMS) fiber (Supelco Co., Bellefonte, PA, USA), previously tested by
Carasek and
Pawliszyn31. The PDMS
fiber was exposed to the volatiles present in the headspace for 30 min
at 37 °C to allow for efficient adsorption and then inserted into the
GC-MS injector for 5 min to achieve desorption of the volatiles.
The volatiles were analyzed using an
Agilent 6890/5975 GC-MS (Agilent Technologies, Santa Clara, CA, USA)
equipped with an HP-5MS capillary column (30m × 0.25mm i.d., 0.25 μm
film thickness). The GC temperature program was as follows: initial
temperature, 40 °C for 5 min; increased to 160 °C at 3 °C/min and held
for 2 min; increased to 250 °C at 8 °C/min and held for 3 min. The
analytical conditions were: flow rate of carrier gas, 1.0 mL/min;
injector temperature, 250 °C;
injection mode, splitless; ion source temperature, 230 °C; quadrupole
temperature, 180 °C; ionization voltage, 70 eV; scanning range,
full-scan mode. The volatiles were authenticated by comparing their MS
spectra with those from the NIST mass spectral library of the GC/MS
system. The relative percentage of the volatiles was measured by area
normalization.
2.8 Nutrient
substances of Qingjinju
seed oils
The total phenolic content of the oil samples was measured using the PRC
National Standards for the grain industry
LS/T
6119-201732.
The
limonin and nomilin content was
determined using high-performance liquid chromatography (HPLC) following
the method described by Chinapongtitiwat et
al.33, with minor
modifications. A measured quantity
of oil samples was mixed with dichloromethane (60 mL) and reflux
extracted at 50 °C for 1 h. The content was then vacuum filtered through
Whatman No. 1 filter paper and concentrated at 50 °C under reduced
pressure using a rotary evaporator (RE-2000B, Shanghai Zengsen
Instrument Technology Co., Ltd, Shanghai, China). The residues were
dissolved in acetonitrile (5 mL) and filtered through a
0.45
μm syringe filter. A portion of the filtered solution (10 μL) was then
analyzed on a C18 HPLC column (4.6 mm × 250 mm, 5 μm) using an Agilent
1260 Infinity II HPLC equipped with a diode array detector (DAD). The
analytical HPLC conditions were as follows: mobile phase,
acetonitrile:water (45:55, v/v); flow rate, 1.0 mL/min; column oven
temperature, 25 °C; DAD detection, 210 nm. The limonin and nomilin
content was quantified by co-chromatography using mixture standards
(Sigma-Aldrich).
The
total α-tocopherol content was
determined using HPLC following the method described by Anwar el
at.15, with
modifications. Oil samples were saponified by mixing the oil (0.5 g)
with 2% ascorbic acid-ethanol solution (100 mL) and 50% aqueous KOH
solution (25 mL) and heating at 70 °C for 30 min. The saponification
liquid was extracted twice with petroleum ether (50 mL) and the combined
organic phases were washed with deionized water (100 mL) until neutral.
The organic layers were filtered through anhydrous sodium sulfate and
evaporated to dryness under a stream of nitrogen. The extracts were
re-dissolved in methanol (10 mL) and
filtered through a 0.22 μm syringe
filter. A portion of the filtered solution (10 μL) was then analyzed on
a C18 HPLC column (4.6 mm × 150 mm, 5 μm) using a Thermo Scientific
Ultimate 3000 HPLC equipped with a diode array-fluorescence detector
(DAD-FLD). The analytical HPLC conditions were as follows: mobile phase,
methanol:water (98:2, v/v); flow rate, 1.0 mL/min; column oven
temperature, 20 °C; excitation/emission wavelengths, 294/328 nm. The
total α-tocopherol content of the samples was quantified by
co-chromatography with pure standards (Sigma-Aldrich).
The total flavonoid content was determined using the method described by
Marinova et al.34, with
modifications. Either oil samples (25 μL) or a standard solution of
rutin was added to a 25 mL volumetric flask and mixed with 5%
NaNO2 (1 mL) for 10 min. A 10%
Al(NO3)3 (1 mL) solution was then added
and the mixture was incubated for an additional 10 min. Finally, 4%
NaOH (10 mL) was added and the volume was brought to 25 mL with ethylene
glycol. The solution was mixed and incubated for 15 min, after which the
absorbance was measured at 510 nm against a prepared reagent blank. The
total flavonoid content of the oils is expressed as mg rutin equivalents
(RE)/100 g oils.
The β-sitosterol content was determined by HPLC using the method
reported by Maria et
al.35