500 g of seeds of Glycine max [Black (S1) and yellow (S3)] and Glycine soja (S2) were collected from different locations across Uttarakhand, West Himalaya, India (Figures 3, 4). The collected seeds were brought to the Rural Technology Complex of GB Pant National Institute of Himalayan Environment (NIHE), Kosi-Katarmal, Almora, situated in the Agro-climatic zone (NARP) Zone-1 with latitude: 29°37′46.9”N, longitude: 79°37′36.3″E, 1160 msL. The experimental site is characterized as maximum temperature ranges between 28 and 29.9°C and the minimum between 11.4 and 20°C with average rainfall of 197.41 mm. Collected genotypes were grown in the same location in the month of May with similar agronomic practices to reduce the effect of the environment on genotypes. Matured seeds were harvested in the second week of October and evaluated for various quality traits. Each seed sample was ground with a mixture grinder (Usha International LTD, India) and sieved to a particle size of 1 mm, which was then used for further analyses. All the chemicals and reagents used in the experimental work were of analytical grade and were brought from HiMedia, Laboratories, Pvt. Ltd., India.

Total ash and fat content were estimated using the Association of Official Analytical Chemists (AOAC) method following Abdulkadir et al. (2016). method. The ash content was calculated by incinerating 2 g of sample in pre-weighed crucibles in a muffle furnace (Macro Scientific Works, India) at 550°C for 5 h. The difference in weight to the initial sample weight was regarded as total ash content. Total fat content was estimated using the Soxhlet method by extracting 2 g of each sample in petroleum ether for 5 h. Carbohydrate and protein content were estimated using a UV–vis spectrophotometer (Hitachi U-2001, Tokyo, Japan) following the Anthrone method by Das et al. (2023) with slight modifications and by Lowry method using the Folin’s reagent (Waterborg, 2009) respectively.

Sample preparation, digestion and analytical determination was done following the method of Kılıç Altun et al. (2017) with slight modifications. One (1 g) of each sample was weighed and transferred to digestion tubes, followed by adding a digestion mixture (2.5 mL HNO₃ and 1 mL H₂O₂). The solution was digested in a closed microwave system (Multiwave Anton Par, 5,000 series) following the procedure: 25°C to 140°C in 15 min then hold for 5 min; 140 to 160°C in 10 min hold for 35 min; then cool to room temperature. After digestion, the solutions were diluted with ultrapure water to 50 mL and mixed. The blank was also prepared likewise. Standard stock solutions (Calcium, Iron, Magnesium, Phosphorus, Potassium, Sodium, and Zinc) were purchased from Merck Pvt. Ltd. India. Identification and quantification of the minerals were carried out by ICP-MS (Agilent, USA based, 7,000 series).

The stock standard solutions of vitamins were prepared by dissolving 10 mg of each standard vitamin purchased from Agilent, weighed, and transferred to a 10 mL volumetric flask. Dissolved and diluted to volume with water as mobile phase (pH adjusted to 2.1 with H₂SO₄). Thereafter, 5.0 g of well-grinded sample was soaked in 20 mL of water (pH 2.1) and kept in the dark for 24 h. The filtrate was then filtered through Whatman No. 1 filter paper, and the volume was adjusted using HPLC grade water. The sample solution was again filtered through a 0.45 μm syringe membrane filter and filled into an auto-sampler vial. After that, the analysis was performed on the HPLC 1100 series Agilent with a DAD detector following the method of Seal (2016). The mobile phase contains water (pH 2.1), the column (300SB-C84.6 × 250 mm, 5 μm pore size) was thermostatically controlled at 25°C, and the injection volume was kept at 20 μL. The total analysis time per sample was kept at 10 min. The flow rate was 1.0 mL/min. HPLC chromatograms of all vitamins were detected using an array UV detector at 210 nm.

Powdered sample (1 g) was extracted via microwave-assisted digestion (Multiwave Anton Par, 5,000 series) in 8 mL nitric acid and 1 mL hydrochloric acid. Then, the samples were diluted with 50% acetonitrile in 1:4 (v/v), and centrifuged at 6,000 RPM for 10 min. The extracts were filtered and dried, followed by the addition of derivatization reagents 0-phthalaldehyde (OPA) and 9-fluorenylmethyl chloroformate (FMOC) to identify total free amino acids. Each derivatization was conducted at 40°C for 10 min and filtered through a 0.45 μm filter before analysis. Amino acid identification was done by Agilent P/N 5061–3,339 high-performance liquid chromatography (Agilent Technologies, USA) following the Agilent method (Mengerink et al., 2002). The separation was performed using gradient elution. Mobile phase A contains 500 mL 20 mM sodium acetate with 2 mg EDTA and 0.018% triethylamine (v/v), and pH adjusted to 7.2 using acetic acid and 0.3% tetrahydrofuran. Mobile phase 2 comprised of 100 mL 20 mM sodium acetate adjusted to pH 7.2 with acetic acid, 200 mL methanol, and 200 mL acetonitrile. A constant flow rate was set at 0.45 mL/min, and the column temperature was set at 40°C. The fluorescence excitation and emission wavelength were set at 338 nm and 390 nm, respectively.

All the parameters were performed in triplicate, and data are expressed as the mean ± standard deviation (SD). Differences within parameters (p < 0.05) were analyzed by the Duncan multiple range test (DMRT) using Statistical Package for the Social Sciences (SPSS) version 29.0.2.0 (20). The principal component analysis (PCA) and heat map analyses were also conducted to correlate phytochemicals, antioxidants, vitamins, minerals, and proximate activity performed in origin software (Origin 2023, Origin Lab, USA).

A well-balanced diet containing adequate amount of all the essential nutrients such as protein, carbohydrates, fat, vitamins and minerals are very essential for proper growth, development, maintenance, and regulation of certain metabolic function in the body (Chen et al., 2018). The proximate composition of soybean samples is shown in Table 1. There was a significant difference (p < 0.05) between the protein content of soybean genotypes. For instance, the highest protein content was recorded in wild soybean (40.98%) followed by black cultivated soybean (37.85%). The highest ash (5.27%) and fat content (22.20%) were observed in yellow cultivated soybean. According to a study by Ren et al. (2012), the ash (4.99%) and fat content (17.86%) was reported highest in yellow cultivated soybean compared to black cultivated soybean having 4.37% fat and 16.64% ash. Similarly, all the samples showed significant differences in carbohydrate content. The highest carbohydrate content was observed in black cultivated soybean (21.75%), followed by wild soybean (20.49%). Etiosa et al. (2017) reported 37.69% protein, 16.31% carbohydrate, and 4.08% ash in soybean seeds. Kim et al. (2023) reported 39.17 to 52.27% protein and 4.74 to 12.68% oil in wild soybean. The protein content in the present investigation was in the range of the reported results (Kim et al., 2023). However, the fat content in the present study was higher than the reported one, and this difference might be due to the difference in extraction solvent, climate, and the other agronomic conditions followed for the genotype’s cultivation. Legumes are mainly consumed because of their richness in protein content and from the results of the present investigation, wild soybean is an excellent source of protein compared to the other soybean genotypes thus, consumption of wild soybean might play an important role in growth, development of muscle mass, maintaining overall health and also in eradication of malnutrition.

Nutrients play a vital role in maintaining the biological structure, regulating metabolic reactions, and properly functioning the immune system (Munteanu and Schwartz, 2022). Soybean genotypes are rich sources of certain minerals, and among them, the highest zinc content was observed in wild soybean (4.68 mg/100 g). The presence of this trace element is effective in treating several chronic diseases such as diabetes, Alzheimer’s disease, Wilson’s disease, and also helps in boosting immunity (Chasapis et al., 2012). The highest magnesium (Mg) (273.00 mg/100 g) and phosphorus (P) (802.00 mg/100 g) content was reported in black cultivated soybean followed by wild soybean. Although black cultivated soybean is a rich source of these minerals, wild soybean also contains greater Mg and P when compared to yellow cultivated soybean. The significant presence of P in black soybeans helps to maintain bone health and Mg aids in DNA synthesis and repair (Hartwig, 2001; Takeda et al., 2012; Takeda et al., 2012). The highest Calcium (301.12 mg/100 g), iron (15.92 mg/100 g dw), and potassium (1785.67 mg/100 g) content was observed in yellow cultivated soybean (Table 1). Whereas, the highest sodium (2.98 mg/100 g) content was observed in black cultivated soybean. It was observed that potassium and phosphorus have statistically strong significant differences amongst the soybean genotypes. The sequence of minerals concentration in soybean genotypes is as follows: potassium>phosphorus>magnesium>calcium>iron>zinc>sodium. Rani et al. (2008) investigated the mineral content in nine varieties of soybean and reported the calcium content ranged from 230.2 to 255.2 mg/100 g, phosphorus in the range 496.1–514.2 mg/100 g, iron content (8.40–11.20 mg/100 g) and zinc content ranged from 7.16 to 7.89 mg/100 g. The results reported in the present study are consistent with the previously reported values, and the mineral content was notably higher than the previously reported values. Very limited data was available on the mineral content estimation in wild soybean. However, an earlier study from 1984 reported higher zinc content in wild soybean(59–83 mg/g) compared to cultivated soybean(38–67 mg/kg) (Raboy et al., 1984). These slight variations in mineral content values are attributable to several factors, including genetic factors, biotic and abiotic influences, genotype, maturity, growing season, geographic location, and agronomic practices (Bellaloui et al., 2015).

Dietary vitamins are essential for human health, normal growth, maintenance, and development (Maqbool et al., 2017). The vitamin content in soybeans is present in very trace quantities (Vineet Kumar et al., 2010). Vitamins such as thiamine, riboflavin, niacin, folate, and pyridoxine were estimated for all three soybean genotypes (Table 2). No significant difference was observed between black cultivated soybean and wild soybean in terms of thiamine and niacin content. Similarly, the level of riboflavin does not show any significant difference between black and yellow cultivated soybean, and pyridoxine showed no significant difference among the soybean genotypes. In summary, there was no significant difference (p > 0.05) observed for vitamins among all the soybean genotypes except for folate. The highest folate (368.00 μg/100 g) content was observed in yellow cultivated soybean followed by wild soybean (356.00 μg/100 g). These vitamins were present in the following order of concentration in soybean genotypes niacin>thiamine>riboflavin>pyridoxine>folate. According to the Food and Nutrition Board, Institute of Medicine the recommended dietary allowance and adequate intake of vitamins for different age groups is presented in Table 2 (NIH, 2024). Based on the recommended dietary allowance, all the soybean genotypes apparently contain an appropriate amount of vitamins necessary for all the age groups. Soybean genotypes contain a significant amount of folate and its consumption is essential for biochemical functions such as DNA synthesis, repair and maintenance (Warzyszynska and Kim, 2014). Humans fail to synthesize folate; thus, it should be taken through dietary sources (Donnelly, 2001). Similarly, niacin is very effective in maintaining cardiovascular health; riboflavin exhibits anticancer, anti-inflammatory, antioxidant, anti-aging, and anti-nociceptive properties; thiamine acts as an important coenzyme for protein, carbohydrates, and pyridoxine acts as a strong anti-inflammatory agent (MacKay et al., 2012; Suwannasom et al., 2020; Mikkelsen et al., 2023; Mrowicka et al., 2023). This is the first study to perform the comparative vitamin estimation among wild and cultivated soybean genotypes.

Amino acids are the building blocks of protein structure and well known to play an important role in protein regulation (Yu and Fukagawa, 2020). A total of 17 amino acids (Figure 5) were analyzed in the soybean genotypes and recorded a significant difference (p < 0.05) amongst most of the amino acids (Supplementary Table S1). It was observed that wild soybean is an excellent source of all the essential and non-essential amino acids. Significant difference was observed in histidine, glutamic acid, glycine, aspartic acid, proline, and phenylalanine among all the samples. Based on the results of the present investigation, glutamic acid is the main component predominantly present in the soybean seeds, followed by aspartic acid and leucine. The concentration of amino acids in soybean genotypes was as follows: Glutamic acid>Aspartic acid>Leucine>Valine>Glycine>Lysine>Phenylalanine>Proline>Isoleucine>Alanine>Tyrosine>Histidine>Cystiene>Threonine>Methionine>Serine>Arginine. All soybean genotypes are rich in essential and non-essential amino acids, possessing various health benefits such as glycine and isoleucine preventing obesity, diabetes and liver diseases (Alves et al., 2019; Yu et al., 2021). Similarly, lysine is a key component of protein synthesis, and glutamic acid is a multifunctional amino acid that helps in promoting brain health. Similar findings of predominance of glutamic acid in soybean genotypes were reported by other researchers (Hong-Tae et al., 2005; Kim et al., 2023). According to a study by Chotekajorn et al. (2021) wild soybean is a great source of various amino acids such as alanine (0.051 mg/g dw), arginine (0.775 mg/g dw), asparagine (0.218 mg/g dw), aspartic acid (0.235 mg/g dw), glutamic acid (0.220 mg/g dw), glycine (0.014 mg/g dw), histidine (0.114 mg/g dw), isoleucine (0.028 mg/g dw), leucine (0.025 mg/g dw), lysine (0.030 mg/g dw), methionine (0.022 mg/g dw), phenylalanine (0.059 mg/g dw), proline (0.022 mg/g dw), etc. The noteworthy number of amino acids in G. soja benefits gastrointestinal development and integrity, enhances immune response potential, influences behavior, and supports sustainability (Meletis and Barker, 2005). Currently, there is a paucity of research focused on the amino acid’s estimation in wild soybean to conduct any comparative study highlighting a critical gap in understanding the amino acid composition of this species.

Consumption of food sources containing significant amount of phytochemicals and antioxidants are of great interest due to their beneficial effects on humans as they offer prevention and protection from certain chronic conditions (Zhang et al., 2015). In the sample context, soybean genotypes evaluated for phytochemical, anthocyanin and antioxidant property showed significant variation (p < 0.05) in all the parameters. Results revealed that the highest total phenol content (TPC) was observed in wild soybean (27.44 mg GAE/g dw) followed by black and yellow cultivated soybeans (15.18 mg GAE/g dw; 14.90 mg GAE/g dw) respectively as shown in Figure 6. The flavonoid content was observed highest in wild soybean (3.319 mg QE/g dw) followed by the other two-colored shades of cultivated soybean. Similar findings were reported by Chen et al. (2021), where the comparative phenol and flavonoid content between wild and cultivated soybeans revealed the highest content in wild soybeans, but the values of the present investigation were somewhat lower than those of the reported one. Additionally, results revealed that black soybeans have significantly higher phenol content when compared to yellow soybeans (Kumari et al., 2015). The highest tannin content was observed in black cultivated soybean (12.03 mg TAE/g dw). Sharma et al. (2013) investigated 20 soybean genotypes and reported the tannin content ranged from 11.1 to 18.8 mg/g dw. The total monomeric anthocyanin (TAC) was found to be highest in black soybeans. The highest anthocyanin content was found in wild soybean (9.150 mg CyE/g dw) followed by black cultivated soybeans (4.79 mg CyE/g /g dw). Yong-Ha et al. (2004) also investigated the total anthocyanin content among thirteen cultivated black soybean cultivars and reported varied anthocyanin ranges from 0.55–16.92 mg/g. Moreover, it was reported in an earlier study that anthocyanins from black soybeans act as a protective agent against oxidative damage to human glial cells (Kim et al., 2012). The antioxidative property of the samples showed that the highest ferric reducing antioxidant power (FRAP) activity was observed to be higher in wild soybean (3.56 mg AAE/g dw) followed by black cultivated soybean (2.62 mg AAE/g dw) and yellow cultivated soybean (1.95 mg AAE/g dw). Likewise, Choi et al. (2021) reported the highest FRAP value in soybeans as 1.58 mg AAE/g dw. The results of the present investigation were somewhat higher, which might be attributed to differences in extraction solvent and specific climatic and agronomic conditions under which the plants were cultivated. It has been previously reported that the genotype, growing environment, and their interaction can significantly impact the phytochemical and antioxidant activities of soybeans (Whent et al., 2009). Several studies have been done to investigate the comparative assessment of the different shades of cultivated soybean, but very limited studies have included wild soybean for the comparative analysis (Zhang et al., 2011; Oh et al., 2019; Choi et al., 2021). The present study has revealed a significant amount of phytochemicals and antioxidants in the wild species and inclusion of this species in the diet may help in the prevention of diabetes, obesity, aging, cancer, cardiovascular and neuro generative diseases (Muscolo et al., 2024).

In this study, heat map analysis examined the correlation between the minerals and vitamins among the different soybean genotypes. It is evident from the heat map that vitamins have a strong positive correlation with minerals. Thiamine was correlated with minerals such as iron, and calcium; riboflavin with sodium; niacin with zinc; folate with potassium, and pyridoxine with iron, potassium, and calcium (Figure 7). Thus, improvement or enhancement of one mineral or vitamin may concurrently enhance the concentration of other minerals and vitamins. Additionally, most of the minerals analyzed showed a strong positive correlation with thiamine and pyridoxine (Figure 5). Similarly, principal component analysis (PCA) was performed to study the relationship between the phytochemicals, antioxidants, nutrients, and the soybean genotypes and between the genotypes themselves. The comparative analysis showed that the first principal component 1 (PC1 = 78.25%) and the second principal component 2 (PC2 = 21.75%) dimensions represent a total of 100% of variations among the parameters and genotypes (Figure 8). It is apparent from the biplot that wild soybean expressed a significant amount of protein, niacin, zinc, anthocyanin, flavonoids, phenol, and antioxidant activity. Black cultivated soybean contains a fair amount of phosphorus, magnesium, carbohydrates, tannin, and protein was found in moderate quantity. Yellow cultivated soybean showed a high amount of thiamine, folate, fat, potassium, sodium, and ash content. In summary, wild soybean and black cultivated soybean contains more Whereas, thiamine, fat, iron, sodium, and riboflavin were present in very less amount in all the three soybean genotypes. Wild soybeans with the highest protein content might be a suitable genetic material for domestication or breeding and, therefore, should be further investigated via advanced biotechnological techniques such as molecular markers for further validation. The biplot shows that fat, iron, calcium, thiamine, pyridoxine, and ash content are positively correlated. Likewise, phosphorus, magnesium, anthocyanin, carbohydrates, tannin, and protein are positively correlated. Phytochemicals, antioxidants, niacin, and protein also showed a strong positive correlation. On the other hand, traits significant in black cultivated soybean and wild soybean showed a negative correlation with the traits significant in yellow cultivated soybean. Additionally, it is also observed that phytochemicals (TPC, TFC, TTC) are negatively correlated with vitamins (Vit B1, Vit B2, and Vit B9) (Supplementary Table S2). This study highlights the advantage of improving traits that showed a positive correlation simultaneously. Whereas the traits that showed negative correlation could be improved independently. The correlation value r ≤ 0.35 characterize a weak correlation, whereas 0.36 to 0.67 signify moderate, and 0.68 to 1.00 shows a strong correlation (Taylor, 1990).