2.1. Plant Seed Materials
The five Fabaceae seeds: Glycine soja (M7), Vigna angularis (M12), Phaseolus lunatus (M9), Phaseolus vulgaris (M14), and Phaseolus coccineus (M10) were collected from Phu Yen, Vietnam in 2018. Samples were stored at −4 °C at the Department of Organic Biochemistry, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology. Samples were botanically identified by Dr. Nguyen Quoc Binh (Department of Biology, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology) and given herbarium numbers.
2.3. Analysis of Fatty Acid, Tocopherol, Sterol Compositions and Phenolic Compounds
The fatty acid composition was determined via gas chromatography and a subsequent ISO draft standard method [
15]. First, 10 mg of extracted oil was added, followed by the introduction of 25 μL of 2M sodium methanolate methanol solution in a vial containing 1 mL of petroleum ether. The closed vial was stirred vigorously for 1 min. Afterwards, centrifugation took place following the addition of 20 μL of water. The aqueous phase was separated and 20 μL of methyl orange in 0.1N HCl was added as a pH indicator. Prior to analysis, the final mixture underwent a thorough stirring. The chromatography instrument for determination of derivatives was a Hewlett-Packard Model 5890 Series II/5989 A, operating in combination with a 0.25 mm ZB-1 fused-silica capillary column (30 m × 0.25 μm i.d.; Phenomenex, Torrance, CA, USA), and a carrier gas of helium at a flow rate of 1.0 mL/min.
To determine tocopherol, HPLC analysis was adopted following a previous procedure [
16]. A mixture containing 250 mg of oil and 25 mL of heptanes was used as the sample for analysis in the HPLC instrument. The instrument was a Merck–Hitachi low-pressure gradient system, equipped with an L-6000 pump and a Merck–Hitachi F-1000 fluorescence spectrophotometer (detector wavelengths at 295 nm for excitation and 330 nm for emission) used in conjunction with Chemstation software. The injection of the sample onto a Diol phase HPLC column (250 mm × 4.6 mm, i.d.; Merck, Darmstadt, Germany) was performed using a Merck 655-A40 autosampler. The flow rate of the column, 1.3 mL/min heptane/tert-butyl methyl ether (99 + 1,
v/
v), was used as the mobile phase. The results were expressed as mg vitamin E/100 g oil.
To determine sterol, the official method of the International Olive Oil Council was employed [
17]. The determination commenced with the saponification of 15 g of oil sample using 50 mL of 2 N ethanolic potassium hydroxide solution. The resultant fraction that was unsaponifiable was added to chloroform, weighed to 20 mg, and applied on a basic silica thin layer chromatography (TLC) plate. The separation of sterol and triterpenediol fraction was performed by eluent mixture with hexane and diethyl ether 65:35 (
v/
v). A UV light was used for the determination of bands following a spraying with 2,7-dichlorofluorescein in 0.2% ethanolic solution, scraping with a spatula, and extraction with chloroform. The extract was then dried and trimethylsilyl ethers were formed by the conversion of sterols and triterpenediols using pyridine hexamethyldisilizane-trimethylchlorosilane (9:3:1,
v/
v/
v). The resultant mixture was allowed to stabilize for 15 min and was subsequently subjected to centrifugation. Sterols were analyzed on a fused silica capillary column coated with 2% isopropanol/98% hexane (30 m × 0.32 mm i.d., film thickness 0.25 μm; Rtx-5: Restek Corporation, Bellefonte, PA, USA; 99 or HP-5: Agilent Technologies Inc., Little Falls, DE, USA). The analysis instrument was a Hewlett Packard series 6890 GC (Waldbronn, Germany) equipped with a split/splitless injector, an autosampler, and a flame ionization detector (FID). First, 1.0 mL of aliquot of 1 derivatized sample solution was split injected into the column (300 °C, split ratio 1:50). Isolation of constituents was performed isothermally at 300 °C, and the determination was carried out with the FID (at 310 °C). Individual peaks of sterols were identified by carrier gas of helium. Relative retention times (RRT) for sterols was determined with reference to a major sterol compound, β-sistosterol, whose RRT is equal to 1 as described by COI [
18].
The total phenolic compound was determined as follows. First, 1 g of powdered seed was ultrasonically extracted with 5 mL of methanol at a concentration of 80% for 30 min at ambient temperature. Then, Whatman Grade 1 filter paper was used to filter the resultant supernatants. The filtrate was stored at 4 °C prior to total phenolic content analysis via the Folin–Ciocalteu method [
19]. Results were expressed as milligram equivalents of gallic acid (GAE) (400–1000 mg/L) per grams of the sample. Average values were reported according to triplicate experimental attempts.
Thirteen phenolic acids were quantified via reversed phase high performance liquid chromatography (RP-HPLC). The instrument used was an Agilent Technologies 1100 series, operating in conjunction with an UV-Vis multiwavelength detector [
20]. The separation was conducted at room temperature with a 250 mm × 4.6 mm, 4 µm Hypersil ODS C18 reversed phase column. Two solvents were used for the mobile phase. The first solvent, denoted as solvent A, consisted of acetonitrile, and the second solvent, denoted as solvent B, was H
2O with 0.2% H
2SO
4. The flow rate was maintained at 0.5 mL min
−1. The progress of the gradient was programmed into six consecutive phases. The duration of phase 1 to 6 was 12, 2, 4, 2, 4, and 4 min, respectively. The ratio of A/B of the solvent system used in phase 1 to 6 was 15/85, 40/60, 60/40, 80/20, 90/10, and 100/0, respectively. The sample for analysis was first subjected to filtration using a 0.45 µm membrane filter and was then injected at a volume of 20 µL. Identification of peaks were made by comparing observations at 280 nm with standards. Triplicate attempts were made for all determinations, in which a specified quantity of trans-2-hydroxycinnamic acid was used as an internal standard.
Antioxidant activity was determined with the DPPH radical scavenging method. For determination of the antioxidant activity of different extracts, the stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used [
21]. This method allows for quick determination of the antiradical power of an antioxidant by measurement of the decrease in the absorbance of DPPH at 515 nm. For each extract, different concentrations were tested. An aliquot (0.5 mL) of the DPPH solution (about 50 mg/100 mL) was diluted in 4.5 mL of methanol, and 0.1 mL of a methanolic solution of the extract was added. The mixture was shaken vigorously and allowed to stand for 45 min in the dark. The decrease in absorbance was measured at 515 nm against a blank (without extract) with a spectrophotometer. From a calibration curve established with different amounts of extract, the ED50 was calculated. The ED50 was the required concentration of an antioxidant to quench 50% of the initial DPPH radicals under the given experimental conditions.
2.4. Composition and Content of Lipid Class Analysis
Total lipid was extracted from fresh seeds by the Bligh and Dyer method [
22]. The seeds (10 g) were crushed, and the lipid was extracted at 4 °C for 24 h by stirring with a mixture of chloroform/methanol (30/60 mL,
v/
v). After that, the mixture was filtered and the biphasic system was obtained by adding chloroform (30 mL) and H
2O (30 mL). After settling (24 h), the lower layer was separated, and the aqueous layer was subject to another extraction with chloroform (30 mL). The total lipid mixture, obtained by mixing and evaporating chloroform layers under low pressure, was stored at −20 °C for further investigation. The total lipid content was determined gravimetrically [
23]. One-dimensional thin layer chromatography (TLC), with pre-coated silica gel plates (10 cm × 10 cm), Sorbfil PTLC-AF-V (Sorbfil, Krasnodar, Russia), was employed to determine total lipid compositions. First, development of the plates took place to full length with
n-hexan/Et
2O/AcOH (70/20/1, by vol.). Following that, the plates were developed to 25% length with CHCl
3/MeOH/C
6H
6/NH
4OH (65/30/5/10, by vol.). The plates were then air dried, sprayed with 10% H
2SO
4/MeOH, and heated at 240 °C for 10 min. To obtain chromatograms, an image scanner (Epson Perfection 2400 PHOTO), operating in a grayscale mode, was utilized. Lipid contents were quantified in percentages using an image analysis program (Sorbfil TLC Videodensitometer, Krasnodar, Russia) with band intensities as inputs. Peak areas and lipid class percentages were calculated according to the previously described method [
24,
25].