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