Phytochemical Profiling of Soybean ( Glycine max (L.) Merr.) Genotypes Using GC-MS Analysis Phytochemical Profiling of Soybean ( Glycine max (L.) Merr.) Genotypes Using GC-MS Analysis

Twenty-four soybean genotypes collected from different regions and origin were evaluated for their quality performance to explore their nutritional and medicinal values. The proxi mate compositions showed considerable variations among soybean genotypes. The USA genotypes recorded the highest values for protein (43.1 g/100 g), total fat (23.61 g/100 g), phenolic content and flavonoids (1.77 and 2.13 mg/g). Using GC-MS analyses of methanolic extracts, a total of 88 compounds were identified in the genotypes and were classified to: 19 heterocyclic compounds, 13 compounds for ketones and esters, 9 for phenolic compound, 7 compounds for carboxylic acids and sugar moiety, 5 compounds for aldehydes and alcohols, 4 ether compounds, 3 amide, 2 alkanes and one alkene and one fatty acid ester. Indonesian genotypes recorded the highest number of phenolic and the Australian genotype A-1 had the maximum number of esters. Genotypes showed high levels of proximate compositions and pharmaceutical components, offering potential candidates for improving those traits in adapted genotypes through breeding program, as well as serving as a good source of mass production of pharmaceutical and medicinal components either through classical or in vitro production. Furthermore, platform was set for isolating and understanding the characteris - tics of each compound for it pharmacological properties.


Introduction
Soybean (Glycine max (L.) Merr) considered among ancient cultivated crops, it was domesticated in the 11th century BC around Northeast of China. It is one of the most widely grown leguminous crops in the world. Its cultivated area was recorded in 95 countries more than 121 million hectare that produced 335 million tons of dry seeds [1] (FAOSTAT, 2016). Soybean had a wide variability, the USDA alone maintains more than 15 thousand soybean accession grouped into 13 maturity classes including both determinate and indeterminate soybean. Early maturing groups are adapted to short summer growing seasons in North USA and Canada while late maturity groups are adapted to southern or coastal plain counties [2]. Soybean occupies an advanced position among agricultural crops, being the most important source of proteins and vegetable oils [3]. Its seeds provided abundant and high quality protein and oil for human diet and animal feed. Its seeds contain more than 36% protein, 30% carbohydrates in addition to fiber, vitamins, and minerals [3]. It also contains about 20% oil, which makes soybean one of the most important edible oil crops. Soy oil has used as binding additives in manufacturing of papers, inks, paints, varnishes, cosmetics, and plastics. It was used also in production of farming pesticides and pharmaceuticals products [4]. Nowadays, biodiesel utilizing soy oil become a new industrial renewable sources of energy. Additionally, soybean as a nitrogen-fixing legume crop helps in reducing the chemical source of nitrogen fertilizers production [4].
Furthermore, tofu, soy milk, soy sauce, miso, etc., have been developed for human consumption, while soya meal (oil extraction by-product) is used as a nutritious animal feed [5]. Moreover, soybean is now regarded as a model legume crop owing to the availability of genome sequence information [6]. Keeping in mind its vast uses, there is huge number of justifications for crop improvement programs throughout the world. Having 53% global production share of all oilseed crops, USA, China, Brazil, Argentina and India gave soybean much attention in the agricultural production systems. Yield and total production of soybean increased over the last two decades due to genetic improvement of this crop [7].
In comparison with conventional legume and animal feed sources, soybean is considered one of the cheapest food resources with medicinal properties due to their highest protein content and no cholesterol due to its contents of Genistein, photochemical and isoflavones [8]. It can help in disease fighting due to its pharmacological properties and its phytochemicals constitutes, including antioxidant, estrogenic, antidiabetic, anti-hypercholesterolemic, antihyperlipidemic, anti-obesity, antihypertensive, anticancer, anti-mutagenic, hepatoprotective, anti-osteoporotic, antiviral, bifidogenic, anti-inflammatory, immunomodulatory, neuroprotective, wound healing, antimicrobial, goitrogenic anti-skin aging, anti-photoaging activity and the effects of anti-nutritional factors [3]. A 111 volatile compounds in fermented soybean curds were reported by Chung [9] and an 83 in commercial plain sufu [10]. Messina [11] reported that the presence of isoflavones in soybean is behind the pharmacological attributes of this crop. Chemical composition included Phenolic acids, flavonoids, isoflavones, saponins, phytosterols and sphingolipids were recorded in soybean [12][13][14]. Due to importance of this crop and its products, this study was amid at estimating the most active constitutes of 24 soybean genotypes including total phenolic, flavonoid and protein content and phytochemicals using GC-MS.

Plant materials
Twenty-four soybean genotypes were grown in Dirab Agriculture Research Station, King Saud University, Riyadh, Kingdom of Saudi Arabia (24_25049.200 N 46_22012.500E) on August, 2014 and were collected from nine countries (Argentina, Australia, China, Egypt, India, Indonesia, USA, and Pakistan). The name and geographical origin of these genotypes are presented in the Table 1.

Proximate composition
Triplicate sample is used to determine the proximate analysis of soybean genotypes for crude proteins, moisture, total ash, fat and carbohydrate by using the methods described in AOAC, [15]. Protein content was estimated using Kjeldahl method with titration and percent nitrogen was determined using [16]

Antioxidants determination
Soybean samples approximately (1 g) were powdered and homogenized in 10 ml 80% methanol. The mixture was shaken at 300 rpm at room temperature for 3 h. Then the extract was centrifuged for 10 min at 3000 rpm and upper aqueous phase were transferred to new Eppndorf tubes. Moreover, the residues were again extracted with 5 ml 80% methanol overnight. The extraction was performed in three replicates, later on extracts combined and stored in dark at 4°C. The Folin-Ciocalteu reagent was used to determine the total phenolic compounds from the extracts using gallic acid calibration curve as standard. The total phenolics were expressed as mg/g gallic acid equivalents (GAE). An extract was aliquot (50 μl) and mixed with Folin-Ciocalteu reagent of 250 μl and 7.5% sodium carbonate of 750 μl. The volume was increased to 5 ml with water and sample was incubated for 2 h. The absorbance was measured at 765 nm against distilled water as blank. The flavonoid determination was measure by aluminum chloride method with the help of Quercetin equivalent as standard. An aliquot of extract (250 μl) was mixed with ddH2O and 5% NaNO 2 (15:1, v/v). After 6 min, 150 μl of 10% AlCl 3 was added to the mixture. A 500 μl of 1 M NaOH was added to the mixture at the 5th min, and volume made up to 2.5 ml with distills water and the absorbance was measured spectrophotometrically at 410 nm.

Gas chromatography-mass spectroscopy
The GC-MS analysis of fractions were performed using a TSQ TM 8000 Evo Triple Quadrupole GC-MS/MS (Thermo Fisher Scientific) equipped with an Elite-5 capillary column (length 30 nm and inner diameter 0.25 mm and film thickness 0.25 μm) and mass detector was operated in electron impact (EI) mode with full scan (50-550 amu). Helium was used as the carrier gas at constant flow rate 1 mL/min and an injection volume of 1 μL. The oven injector temperature was programmed from 50°C with an increase of 8°C/min to 200°C, then 7°C/min to 290°C/min. The results were compared using the database of National Institute Standard and Technology (NIST).

Data analysis
The data were subjected to descriptive statistics (mean, standard deviation, coefficient of variability, minimum and maximum values) and principal component analysis (PCA) using statistical software Past3 program [17].

Proximate analysis
The proximate analysis values of 24 soybean genotypes (crude protein, ash fat, carbohydrate, and moisture contents) values and total phenolic and flavonoid contents are shown in Table 2, and the detailed proximate analysis estimates are presented in Table S1. The minimum crude protein value was recorded for Argentinian (35.63%), while maximum recorded for Clark genotypes (43.13%). The genotypes, i.e., Clark, Indo-1, Indo-black, Ijen, Romal-1, X 30 and 3803 recorded higher than 40% crude protein. The significant variations for crude proteins among genotypes were recorded and that might observed due to differences in genetic background and/or origin. The higher protein content in the genotypes is also reported previously which ranged from 43 to 45% [18]. These results are also in line with Zarkadas et al. [19,20] who reported crude protein contents in soybean ranging from 33.67 to 42.11%. The minimum moisture contents were recorded in Giza 83 (3.08%) while maximum was recorded for Indo-1 (5.88%) with an average (4.90%) mean value showing non-significant difference. Ash contents ranged from 4.55 to 6.28% with an average of 5.44%. The maximum was recorded for Giza 111 (6.28%) genotype while Romal-1 genotype had the lowest (4.55%) of ash contents. The moisture and ash contents values were recorded lower than that reported by [21]. Total fat ranged from 16.92 to 22.94% with a mean value of 21.16%. The genotype Indo-black contained the lowest while the genotype 3803 recorded the highest content. Soybean is considered about 47% of its energy value in fat content [22,23] which is compared to other legumes. Our results regarding total fat were in line with that of [24] who reported that that total fat value ranged 18 and 22 g/100 g in soybean genotypes. The minimum carbohydrate content in Clark (26.11%) while maximum in Argentinian (33.18%), with an average (29.48%) was recorded among soybean genotypes.

Flavonoid and phenolic contents
Flavonoid and phenolic compounds are the important phytochemicals and natural antioxidants founds in fruits, vegetable and cereals grains. It serves as multiple biological functions, i.e., defense against cardiovascular disease, cancer and aging [25]. The results regarding total phenolic and flavonoids contents for 24 soybean genotypes are presented in Table S1, and significant differences were recorded for all soybean genotypes. The seed extracted results indicated that the maximum phenolic contents was recorded in Romal-1 (1.7 mg/g) while minimum in Giza 111 (1.15 mg/g) with an average 1.45 GAE/g mg/g ( Table 2). However, total flavonoid content ranged 0.68 to 2.13 mg QE/g ( Table 2). Phenolic content is strongly linked with antioxidant capacity [26,27] and can contribute towards antioxidants activities [28]. The use and demands of phenolic are increasing rapidly in food industry to enhance nutritional value and quality of food [29].

Principal component analysis (PCA)
The first three principal components explained 78.64% of total variations among genotypes ( Table 4 and Figure 3).  Table 4. Eigen values and proportion of the variance explained for the three principal components of the 24 soybean genotypes based on phytochemical components. and permitted soybean researchers to more easily develop significant relationships between important soybean characteristics. Soybean cultivars have been classified using (PCA) of the fatty acid data [41]. The first four principal components generated in total 81.49% of the variance, where PC1 positively correlated with oleic, linoleic, and gondoic acids, PC2 with stearic, linolenic and arachidic acids, PC3 behenic and lignoceric acids, and PC4 by palmitic acid. Moreover, due to the ability of PCA to manage and interpret large data sets, it has been used in studying relationships that exist in fatty acid characterization [42]. Although soybean oil has been included in some chemometric studies comparing vegetable oils, soybean cultivars have yet to be extensively classified using multivariate techniques [43,44].

Conclusion
The results revealed that soybean genotypes cover variable patterns of total proteins flavonoids, phenolic and various bioactive volatile compounds. The mass spectrometry analysis results demonstrated that, majority of soybean genotypes are a source bioactive compounds with antioxidant, antiinflammatory, antimicrobial and other functions. 2-Methoxy-4-vinylphenol, phenol, 2,6-dimethoxy-, 3,5-dimethoxyacetophenone, hexadecanoic acid methyl ester, 1,2-cyclopentanedione, and 3,5-dihy droxy-6-methyl-2,3-dihydro-4H-pyran-4-one were present in majority of genotypes. However, the genotypes Ijen and Indo-1 contributed more phenolic compound than others genotype. Genotype A-1 has the maximum compound in esters compounds. The genotypes Indo-11 and 3803 contribute maximum ketone compounds while Giza 111 contributes more in heterocyclic compounds. Some genotypes may have good therapeutic potential and could be served as a potential source in drug wdevelopment as a health supplement. This study also provides a platform for isolating and understanding the properties of each compound for it pharmacological properties.