Effects of Salt Stimulation on Lunasin Accumulation and Activity during Soybean Germination

Lunasin, a bioactive peptide, was originally found in soybeans, and it has exhibited multiple biological functions. On the basis of previous studies, salt stress was found able to induce changes in many polypeptides and translatable mRNA levels in plants. Salt stress was applied to soybean germination, with water treatment as a control group, to evaluate the effects of salt stimulation on lunasin accumulation and activity during soybean germination. Lunasin content gradually increased in the control group during germination, reached the highest level after six hours of imbibition, and then slowly decreased. Under salt exposure, lunasin content showed a similar trend to that of the control group. The lunasin content in salt-treated soybean was significantly higher than that in the control group. Lunasin peptide was purified from soybean after six hours of imbibition and it was then used for function evaluation. Purified lunasin from salt-stress-germinated soybean (6 h-LSGS) exhibited stronger antioxidant activity than lunasin from water-treatment-germinated soybean (6 h-LWGS) and soybean seed without imbibition (DRY). The 6 h-LSGS presented anti-inflammatory activity on LPS-induced macrophage cells (p < 0.05) by suppressing the release of nitric oxide (NO) and proinflammatory cytokines, including IL-1 and IL-6. The gene expression of NOS, IL-1, IL-6, and TNF-α was significantly inhibited by 6 h-LSGS. Further, 6 h-LSGS exhibited superior antiproliferation activity on human breast-cancer cells MDA-MB-231 when compared to 6 h-LWGS and DRY. Overall, this study offers a feasible elicitation strategy for enhancing lunasin accumulation and its properties in soybean for possible use in functional food.


Introduction
Lunasin, which is a soybean-derived bioactive peptide, has shown positive effects on many biological functions. Lunasin, originally discovered in soybeans, has a molecular weight of 5.5 KD million and 44 amino acids. Arg, Gly, and Asp residues and the cell-adhesion module that is composed of nine aspartic acid residues at the carboxyl terminal determine the biological activity of lunasin. Lunasin reaches the target organ or tissue through decomposition by the gastrointestinal digestive enzyme, serum protease, and peptidase in the body. Subsequently, it binds to cells through Arg-Gly-Asp, and regulates cell migration, growth, differentiation, and apoptosis [1]. Extensive scientific research has shown that lunasin has natural antioxidant, antiallergic, and anticancer effects, and it helps to regulate cholesterol biosynthesis in vivo [2].
Germination is one of the ways to promote the significant accumulation of lunasin in soybeans [3]. In the process of soybean germination, temperature, germination time, and light treatment have been affected to accumulate lunasin [4,5]. Salt stress is considered to effectively promote secondary metabolic biosynthesis in plants, such as phenolic compounds, saponins, alkaloids, and gluconate [6,7]. Previous research showed that salt stress could induce changes in many polypeptides and translatable mRNA levels in plants. Studies on NaCl treatment length, NaCl concentration, salt-stress recovery, and the effects of other stresses showed that these peptides play a special role in plant salt stress [8].
Seed germination refers to a series of orderly physiological and morphogenetic seed processes, starting from imbibition. Sometimes, NaCl stress does not induce polypeptides to disappear, or cause the synthesis of unique polypeptides, but it could decrease or increase the synthesis of a number of polypeptides [9]. Félicie et al. compared the patterns of total protein that were extracted from leaves of control and salt-treated plants, and found that a 22 kDa, pI 7.5 polypeptide accumulated when plants were exposed to NaCl [10]. Asian countries have the traditional habit of eating soybean sprouts, and its composition change is of great significance in food processing and consumption. This study not only evaluates the effects of salt stimulation on lunasin content and activity in the soybean germination process, but it also identifies the key germination time point of greater lunasin accumulation by salt stimulation. It was the first time that salt stress was applied to soybean germination, which aimed at increasing lunasin content and activity.

Soybean Germination
The soybeans were subjected to two different treatments: germination under water treatment or under salt stress. The seeds were disinfected by immersing in 1% sodium hypochlorite for five min. and rinsing repeatedly with distilled water. Washed high-quality soybean seeds that were the same size and without decolorization were soaked in 25 • C water or 50 mM NaCl solution for 1 h, and then transferred into Petri dishes placed in a thermostat dark house. Germination was carried out in dark conditions at 25 ± 1 • C. Soybean sprouts in each group were collected after 6, 12, 24, 36, and 48 h, and immediately stored at −80 • C.

Protein Extraction and Purification
Phosphate-buffered saline (PBS) was used as extraction solution; the ratio of material to liquid was 1:10 and extraction was at 4 • C for 48 h. The supernatant was obtained by centrifugation. Peptides with a molecular weight of less than 10 KD were obtained by membrane ultrafiltration [11]. We referred to Ren's method with minor modification for further low-molecular-weight peptide purification [2].

Western Blot and ELISA Detection
Western blot analysis was performed on the basis of a previously published method [12], with minor modifications. The protein sample that was separated by SDS-PAGE (sodium dodecyl sulfatepolyacrylamide gel electropheresis) was transferred to the solid-phase carrier (nitrocellulose film). After rinsing the membrane with PBS for 10 min., the membrane was moved to the 5% skimmed-milk-powder sealing solution that was configured with PBST, and shaken and sealed in the shaker at room temperature for 2 h. The first antibody was diluted with tris-buffered saline containing 0.05% Tween-20 (TBST) to an appropriate concentration, and the membrane was removed from the blocking solution and then placed in the antibody diluent. The shaker was incubated overnight at 4 • C. It was then washed with TBST in a shaker at room temperature three times for 10 min. each time. The diluent of the second antibody was prepared with the same method and it came into contact with the membrane and incubated at room temperature for 2 h. The chemiluminescence reaction was carried out by washing with TBST in a shaker at room temperature three times for 10 min. each time [13].
The ELISA refers to the Dia method, with minor modifications [14]. The samples were diluted to working concentration with coated buffer, cultured at 37 • C for 3 h, removed the coating solution, and washed four times. The pat protein solutions of different concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, 50, and 100 µg/L) were added to the enzyme plate, each concentration was repeated three times, incubated at 37°C for 0.5 h, and the plate was washed four times. Subsequently, 100 µL McAb solution was added, diluted to the working concentration, cultured at 37 • C for 0.5 h, and the plate was washed four times. After washing the plate, enzyme-labeled sheep antirabbit antibody, 1000-fold diluted, was added and incubated at 37 • C for 0.5 h four times. Finally, 100 µL substrate buffer was added to each well; after 10 min., 50 µL terminating solution was added to each well to measure the OD value of each well at 450 nm wavelength.

Antioxidant-Activity Determination
The scavenging rates of DPPH and ABTS + free radicals were determined with reference to the method in [15][16][17][18], with minor modifications. For DPPH analysis, DPPH was dissolved in methanol, and samples were prepared in a solution with a concentration of 0.125/0.25/0.5/1 mg·mL −1 . We then placed 2 mL DPPH in the test tube, added 2 mL sample solution, mixed well, avoided light for 1 h, and then determined sample absorbance at 517 nm. For ABTS + analysis, samples were dissolved in ultrapure water, and the final concentration gradient was 0.125/0.25/0.5/1 mg·mL −1 . We put the ABTS + solution in the test tube, added the sample solution, mixed well, avoided light for 2 h, and then determined the absorbance of the sample at 734 nm [18]. The antioxidant properties of ABTS + radicals are different from those of DPPH. ABTS analysis is superior to DPPH analysis when the sample contains hydrophilic antioxidants [19].

Anti-Inflammatory-Activity Assay
The experiment was carried out according to the Dia scheme [14]. After anti-inflammatory-activity assay, the RAW264.7 cell was collected and used for RNA isolation on the basis of the protocol of the Cell RNA Extraction Kit (TianGen Biotech, Beijing, China); cDNA was synthesized while using a cDNA Synthesis SuperMix Kit (TianGen Biotech, Beijing, China). NOS, IL-1, IL-6, and TNF-α expressions in the RAW264.7 cell were measured through qRT-PCR, which was performed on an ABI 7500 Real-Time System (Applied Biosystems, San Francisco, CA, USA). The mouse actin gene was used as the control to calculate gene expression in the qPCR according to the 2 -∆∆Ct method. Table 1 shows all of the primers. Table 1. Primers and sequences.

Anticancer-Activity Assay
MDA-MB-231 cells were incubated in a DMEM medium that was supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum, and filled with 5% CO 2 cells at 37°C. The cells were electroplated in 96 well plates at 2 × 10 4 cell/hole density for overnight incubation, and treated with lunasin at different concentrations and incubated for 72 h. Afterwards, Hank's Balanced Salt Solution (HBBS) was added to the cells and placed at 37°C for 1 h. Subsequently, absorbance at 570 nm was calculated by spectrophotometer.

Statistical Analysis
All of the experiments were repeated more than three times. The values are expressed as the means of three independent experimenters' SD (STDEV). GraphPad 5.0 (GraphPad Software Inc., San Diego, CA, USA) and SPSS 17.0 (SPSS Inc., Chicago, IL, USA) were used for statistical analysis. The difference was statistically significant (* p < 0.05, ** p < 0.01). SPSS analyzed all of the graphical representations. Figure 1 shows the expression patterns of lunasin at different soybean germination stages. During the germination of soybean seeds, lunasin bands significantly deepened in hours 0-6, peaking at 6 h, and obviously decreasing thereafter ( Figure 1A). Under salt exposure ( Figure 1A), the lunasin bands showed similar patterns and were significantly increased in depth compared to the control. This suggests that lunasin content was significantly accumulated after salt treatment, which indicated that it was viable for increasing the content of lunasin in soybean by the salt treatment of the germinating soybeans.

Lunasin-Content Detection
Lunasin content was measured through ELISA ( Figure 1B). The contents of the lunasin peptide in the soybeans were 0. stress could be due to altered mRNA processing, transcription regulation, transport, stability, or due to the changed rates of protein degradation [8]. It might also be due to the inhibition or stimulation of mRNA translation to varying degrees by increased cytoplasmic ion (Na and Cl) concentrations [20]. Park et al. found that the lunasin content accumulated during soybean germination, similar to a previous study [3]. Paucar-Menacho et al. also showed that lunasin content increased by 61.7% during soybean germination at 25°C for 42 h [21].

Mass Spectrometry Analysis
UPLC-MS/MS was used to further confirm that lunasin was indeed present in the sample and the ELISA results. The lunasin chromatograms clearly showed a peak at the retention time of 3.66 min. (Figure 2A) Figure 2B), which was consistent with a previous report [22].

Mass Spectrometry Analysis
UPLC-MS/MS was used to further confirm that lunasin was indeed present in the sample and the ELISA results. The lunasin chromatograms clearly showed a peak at the retention time of 3.66 min. (Figure 2A). The mass spectrum acquired from the peak at 3.  Figure 2A indicate that the peak area above 2.0e6 will be displayed. The arrow in Figure 2B indicate that ion fragments with a strength of more than 8000 will be displayed.
Lunasin can protect Caco-2 cells from oxidative damage caused by hydrogen peroxide and tert-butyl hydroperoxide, similar to the results of our research [23]. These findings confirm that lunasin has effective antioxidant activity. In addition, it was previously shown that lunasin can inhibit experiment cataract induced by d-galactose in rats and upregulate antioxidant enzymes [24]. Ren et al. in vitro studied lunasin antioxidant activity in quinoa [2]. The superior antioxidant effect of the lunasin extract from salt-treated soybean in comparison to that from the control could be ascribed to the higher accumulation of lunasin. However, as salt stress can induce the accumulation of a variety of antioxidants, such as saponins, isoflavones, tocopherols, and carotenoids [25], the synergistic effect of these antioxidant components might contribute to the significant increase in antioxidant activity of salt-stress samples [26].

Anti-Inflammatory-Activity Assay
In this study, the anti-inflammatory function of lunasin was evaluated through monitoring the immune response in LPS-stimulated mouse macrophage 264.7 cells. In the cytotoxicity assay ( Figure 4A), after 24 h incubation with 6 h-LSGS, 6 h-LWGS, and DRY, the survival rate of cells did not change much at the tested concentrations (0.25-2 mg·mL −1 ). In the NO assay ( Figure 4B), 6 h-LSGS, 6 h-LWGS, and DRY significantly inhibited NO accumulation in the culture medium in a dose-dependent way. A significant increase was noted in the inhibition rate of 6 h-LSGS (70.2%) at 2 mg·mL -1 in comparison with 6 h-LWGS (58.47%) and DRY (51.97%). Further, the gene expressions of NOS, IL-1, IL-6, and TNF-α were significantly upregulated after LPS induction, as shown in Figure 4C. However, expression levels were highly inhibited by lunasin extract from the soybean. In addition, 6 h-LSGS exhibited a significantly higher inhibition rate than that from 6 h-LWGS. The better anti-inflammatory effect of salt stress in comparison to that of the control could be the result of increasing lunasin content in soybean sprouts after salt treatment. The results suggested that soybean lunasin could inhibit NO accumulation, as well as gene expressions either directly or indirectly associated with inflammation.
Similar to our study, lunasin was reported to inhibit NOS expression in LPS-induced RAW264.7 macrophage cells, which suggested that lunasin performed its anti-inflammatory activity by regulating the iNOS/NO signal pathway [14]. Blanca et al. investigated the anti-inflammatory activity of lunasin on the mouse macrophage 264.7 cell line, and macrophage cells were not inhibited by lunasin-related fragments, and found that the complete primary sequence of lunasin was needed to reduce the reactive oxygen species (ROS) induced by LPS-induced macrophages [27]. Further, Cam and De Mejia found that lunasin has the potential to inhibit αVβ3 integrin-mediated proinflammatory markers by downregulating the activation of the Akt-mediated NF-κB pathways [28].

Anti-MDA-MB-231 Activity Analysis
The effects of lunasin on chemical carcinogens were confirmed in cells in vitro [29,30]. Moreover, lunasin can inhibit the transformation of mammalian cells induced by oncogene E1A, and reduce cancer incidence in mouse models [30]. This study measured the cytotoxic and antiproliferation effects of lunasin purified from soybeans on human breast-cancer MDA-MB-231 cells. The cell cytotoxic assay results showed that lunasin from DRY, 6 h-LWGS, and 6 h-LSGS presented no cytotoxicity to MDA MB-231 cells at concentrations from 0.5 to 2 mg·mL −1 ( Figure 5A). In the antiproliferation assay ( Figure 5B), MDA-MB-231 cell proliferation was highly inhibited by DRY, 6 h-LWGS, and 6 h-LSGS. In addition, 6 h-LSGS (69%) exhibited a significantly higher inhibition rate on cell proliferation than did 6 h-LWGS (52%) and DRY (37%) at a concentration of 2 mg·mL −1 . Consistent with our research, Jiang et al. found that lunasin can inhibit the proliferation and differentiation of breast-cancer cells [1]. Lunasin suppressed the metastasis of breast-cancer cells through the inhibition of the NF-κB and FAK/Akt/ERK signaling pathways. Furthermore, Hsieh et al. demonstrated that human estrogen-independent breast-cancer MDA-MB-231 cells are significantly inhibited by lunasin when combined with aspirin when compared with inhibitions after using each compound alone [31].

Conclusions
This paper evaluated the effects of salt stimulation on lunasin accumulation and activity during soybean germination; 50 mM NaCl was applied to soybean germination, and water treatment was recognized as the control group. The lunasin content gradually increased in the control group during germination, reached the highest level after 6 h imbibition, and then slowly decreased. Lunasin content also exhibited a trend of increasing and then decreasing under salt exposure. The lunasin content in germinating soybeans under salt stimulation was significantly higher than that in the control, and salt stimulation (6 h-LSGS) was 2.5 times that of the control group (6 h-LWGS). Moreover, 6 h-LSGS exhibited stronger antioxidant, anti-inflammatory, and anticancer activity than 6 h-LWGS. Overall, this study offers a feasible elicitation strategy for enhancing lunasin accumulation and its properties in soybeans for possible use in functional food.

Conflicts of Interest:
The authors declare no conflict of interest.