Potential Health Benefits Associated with Lunasin Concentration in Dietary Supplements and Lunasin-Enriched Soy Extract

Lunasin has demonstrated antioxidative, anti-inflammatory, and chemopreventive properties. The objectives were to evaluate the concentration of lunasin in different lunasin-based commercial dietary supplements, to produce a lunasin-enriched soy extract (LESE) using a two-step pilot-plant-based ultrafiltration process, and to evaluate their biological potential in vitro. LESE was produced using 30 and 1 kDa membranes in a custom-made ultrafiltration skid. Lunasin was quantified in eight products and LESE. Lunasin concentrations of the lunasin-based products ranged from 9.2 ± 0.6 to 25.7 ± 1.1 mg lunasin/g protein. The LESE extract contained 58.2 mg lunasin/g protein, up to 6.3-fold higher lunasin enrichment than lunasin-based dietary supplements. Antioxidant capacity ranged from 121.5 mmol Trolox equivalents (TE)/g in Now® Kids to 354.4 mmol TE/g in LESE. Histone acetyltransferase (HAT) inhibition ranged from 5.3% on Soy Sentials® to 38.3% on synthetic lunasin. ORAC and lunasin concentrations were positively correlated, and HAT and lunasin concentrations were negatively correlated (p < 0.05). Melanoma B16-F10 and A375 cells treated with lunasin showed dose-dependent inhibitory potential (IC50 equivalent to 330 and 370 μM lunasin, respectively). Lunasin showed protein kinase B expression (57 ± 14%) compared to the control (100%) in B16-F10. Lunasin concentration found in commercial products and lunasin-enriched soy extract could exert benefits to consumers.


Introduction
Lunasin is a forty-three amino acid peptide originally isolated from soybeans. It features a unique amino acid sequence containing an arginine-glycine-aspartic acid cell adhesion motif and a polyaspartic acid tail on its carboxylic acid end [1][2][3][4][5][6][7][8][9][10][11][12]. The internalization of lunasin in macrophages is primarily mediated by endocytic mechanisms that involve integrin signaling, clathrin-coated structures, and micropinosomes. The aggregation of the clathrin-coated structures and the punctate localization of lunasin at the intracellular sites could indicate lunasin endocytosis and internalization into the nucleus via nucleolar sequestration [13].

Pilot-Plant Production of Lunasin-Enriched Soy Extract
Production of lunasin-enriched soy extract was performed in the pilot plant of the Center for Crops Utilization Research, Iowa State University (Ames, IA, USA). Defatted soy flour was suspended in deionized water in a 1:10 ratio and mixed for 90 min at room temperature. After that, the mixture was centrifuged with a Centrisys horizontal decanter (Model no. CS10-4 3PH, Kenosha, WI, USA) at 4255 rpm bowl speed, 2 rpm differential solid scroll speed, with 11.5 L/min (LPM) feed speed. The resulting supernatant was subjected to two-stage ultrafiltration using membranes with 30 kDa and 1 kDa molecular weight cut-offs obtained from Sepro Membranes, Inc. (Oceanside, CA, USA). The pilot-plant ultrafiltration system was a custom-made skid manufactured by MP&C, Inc. (Edgar, WI, USA). The ultrafiltration was carried out using the 30 kDa cartridge under the following conditions: a feed rate of 13 LPM, a recirculation pump outlet pressure of 30 psi, and a permeate flow rate of 1.6 LPM by adjusting the retentate/permeate ratio control valve and the recirculation pump and the high-pressure pump. The permeate containing molecules with molecular weight <30 kDa were collected and further subjected to another ultrafiltration step using 1 kDa molecular weight cut-off membrane with the following conditions: a feed rate of 10 LPM, a recirculation pump outlet pressure of 100 psi, and a permeate flow rate of 1.2 LPM by adjusting the retentate/permeate ratio control valve and the recirculation pump and the high-pressure pump. The concentrated retentate was collected and freeze-dried as the final enriched lunasin extract.

Analysis of Lunasin during Pilot-Plant Based Production of Lunasin-Enriched Soy Extract
Samples of the decanter supernatant, the retentates and permeates from both 30 and 1 kDa membrane treatments were analyzed for lunasin using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis as previously reported [1].

Lunasin Purification Anion Exchange Chromatography
Lunasin extract (LE) was obtained as reported previously [26] with slight changes. Briefly, freeze-dried lunasin enriched soy extract (40% w/v) was solubilized in 50 mL distilled water, centrifuged twice at 12,000× g for 10 min, and filtered through a 0.45 µm filter. A HiTrap Q HP (GE Healthcare Bio-Sciences, Uppsala, Sweden) column was used coupled with a HiPrepp 26/10 desalting pre-column (GE Healthcare Bio-Sciences, Uppsala, Sweden). Unbound proteins eluted with Tris-HCl 20 mM, pH 7.4 at a 1 mL/min flow rate. Bound proteins eluted using 0.4 M NaCl. The LE sample was desalted using ultrafiltration through a 1 kDa disc and freeze-dried for in vitro analysis.

Protein Concentration Measurement by Detergent-Compatible (DC) Protein Assay
The protein concentration of lunasin-based commercial products and samples from ultrafiltration production of lunasin-enriched flour were determined by microplate DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA) as previously reported [1] and calculated using a bovine serum albumin (BSA) standard curve (y = 0.0003x + 0.0209, R 2 = 0.99).

Enzyme-Linked Immunosorbent Assay
Lunasin concentrations of lunasin-based commercial products and samples from ultrafiltration production of lunasin-enriched soy extract were determined by ELISA as previously reported [1] and calculated using a synthetic lunasin standard curve (y = 0.0076x − 0.1902, R 2 = 0.96).

Antioxidant Capacity
The antioxidant capacities of all the samples were measured by the oxygen radical absorbance capacity (ORAC) assay as previously described [28] and the results were expressed as mmol Trolox equivalents (TE)/g and calculated using the generated Trolox standard curve (y = 0.1150x − 3.17, R 2 = 0.98).

Histone Acetyltransferase (HAT) Inhibitory Screening Assay
HAT inhibition assay was performed according to the manufacturer protocol (Cayman Chemicals, Ann Arbor, MI, USA). The assay was conducted as follows: 15 µL of assay buffer, 5 µL of acetyl CoA, 10 µL of diluted HAT/pCAF, and 5 µL of diluent or samples (final concentration of 100 µg protein/mL for samples and 10 µM for synthetic lunasin) were added to wells in triplicate. The reactions were initiated by adding 20 µL of HAT peptide except for background wells and incubated in a shaker for 5 min at room temperature. After incubation, the reaction was stopped by adding 50 µL of HAT stop reagent to all wells and 20 µL of HAT peptide was added to the background wells. After which, 100 µL of HAT developer was added to each well followed by incubating for 20 min at room temperature. The plates were read using a Synergy 2 microplate reader (Winooski, VT, USA) at 340/30 nm excitation wavelength and 440/40 nm emission wavelength. Inhibition of HAT was calculated based on the total HAT initial activity well (treated with diluent only).

Protein Kinase B (Akt) Pathway Expression
Cells were treated with lunasin half-maximum inhibitory concentration (IC 50 ) for 24 h, and then cell lysates were produced. The protein concentration was quantified with Bio-Rad DC protein assay. Based on preliminary results obtained using Mitogen-Activated Protein Kinases (MAPK) (ab211061; Abcam, Cambridge, MA, USA) array (data not shown), Protein kinase B (Akt) (AAH-AK T-1-8; Ray Biotech, Norcross, GA, USA) was focused on using a protein concentration of 300 µg of protein/mL. The epitopes for AKT array are shown in Table 2.

Statistical Analysis
All analyses were performed in at least two independent replicates with two replicates. Data were analyzed using the proc GLM command in SAS software version 9.4. Statistical significance was reported at p < 0.05 and Tukey posthoc test was applied. GraphPad Prism 8 was used for IC 50 analysis. AKT membranes were analyzed using a t-test. Table 1 presents lunasin concentrations while Figure 1 shows the protein profile (a) and western blot analysis (b) of different commercial lunasin-based dietary supplements. On a serving basis, LunaRich X™ capsule had the lowest lunasin concentration, 0.2 ± 0.0 mg lunasin/serving (41.0 ± 2.1 mg lunasin/100 g powder), while Now ® kids-vanilla presented the highest lunasin concentration, 7.6 ± 0.3 mg lunasin/serving (26.2 ± 1.1 mg lunasin/100 g powder) ( Table 1). Figure 1a shows that all the lunasin-based products contained mixtures of different soy proteins, indicating a simple extraction and/or purification steps during preparation. The protein profiles of the eight products contain the major proteins found in soybeans as previously reported [29]. Figure 1b confirms the presence of lunasin in the commercial products as they show a positive reactivity towards lunasin rabbit polyclonal antibody. Studies on the concentration of lunasin in commercial soy products have been previously reported. No studies, however, evaluated lunasin concentrations in lunasin-based dietary supplements.

Statistical Analysis
All analyses were performed in at least two independent replicates with two replicates. Data were analyzed using the proc GLM command in SAS software version 9.4. Statistical significance was reported at p < 0.05 and Tukey posthoc test was applied. GraphPad Prism 8 was used for IC50 analysis. AKT membranes were analyzed using a ttest. Table 1 presents lunasin concentrations while Figure 1 shows the protein profile (a) and western blot analysis (b) of different commercial lunasin-based dietary supplements. On a serving basis, LunaRich X™ capsule had the lowest lunasin concentration, 0.2 ± 0.0 mg lunasin/serving (41.0 ± 2.1 mg lunasin/100 g powder), while Now ® kids-vanilla presented the highest lunasin concentration, 7.6 ± 0.3 mg lunasin/serving (26.2 ± 1.1 mg lunasin/100 g powder) ( Table 1). Figure 1a shows that all the lunasin-based products contained mixtures of different soy proteins, indicating a simple extraction and/or purification steps during preparation. The protein profiles of the eight products contain the major proteins found in soybeans as previously reported [29]. Figure 1b confirms the presence of lunasin in the commercial products as they show a positive reactivity towards lunasin rabbit polyclonal antibody. Studies on the concentration of lunasin in commercial soy products have been previously reported. No studies, however, evaluated lunasin concentrations in lunasin-based dietary supplements. Cavazos et al. [26] reported that lunasin concentrations ranged from 1.6 to 22.2 mg lunasin per serving in different organic soymilks, soy protein shakes, and soy infant formulas. Hernandez-Ledesma et al. [30] reported that different soymilks had lunasin concentrations of 10.7 to 18.9 mg/100 mL of milk, which can be translated from 25.7 to 45.4 mg lunasin per 240 mL serving. Our results showed that the lunasin concentrations of lunasin-based products were 0.2 ± 0.0 to 7.6 ± 0.3 mg lunasin/serving (240 mL), which typically falls within the standard lunasin concentrations that can be found in different soymilk and soy-based infant formulas. Table 1 shows the lunasin concentration expressed as mg lunasin/100 g product and mg lunasin/serving, as well as serving size and ingredients that can be found in each commercial lunasin-based dietary supplement. Previous in vitro studies have shown that as little as 10 µM of lunasin led to reducing pro-inflammatory markers TNF-α, IL-6, and IL-1β in lipopolysaccharide-induced macrophages [14,25]. Considering the low bioavailability of lunasin and a total blood volume of five L, approximately 250 mg of lunasin is needed to reach the 10 µM lunasin concentration. Based on the lunasin concentrations in different lunasin-based commercial dietary supplements, approximately 33 servings (Now ® Kids-Vanilla) to 1250 servings (LunaRich X™ capsule) would be needed to reach this concentration. These estimates assume that lunasin is 100% bioavailable. Previous studies on the bioavailability and digestibility of lunasin showed that 97% of lunasin is digested after pepsin-pancreatin digestion [31] and only 4.5% of the remaining lunasin can be absorbed [32] indicating that a larger number of servings of these products is needed in order to reach an effective concentration of 10 µM. Continuous intake of soy-based products, however, may lead to sustained lunasin concentrations in plasma, which might be responsible for health benefits associated with soy consumption. Figure 2a,b show the soluble protein and lunasin concentrations of soy materials at different stages of production of lunasin-enriched soy extract, respectively. The protein concentration of the starting supernatant after centrifuging the defatted soy flour:water mixture (1:10 ratio) was 42.6 mg/mL. After 30 kDa ultrafiltration, the retentate had an increased protein concentration to 62.5 mg/mL, which is expected as molecules <30 kDa passed through the 30 kDa membrane cartridge. The increase in total soluble protein can also be attributed to the concentration of the starting supernatant to 30 kDa retentate. On the other hand, the protein concentration of the 30 kDa permeate was 3.5 mg/mL, at least 12x lower than the starting supernatant. This could be attributed to dilution of this fraction as more water passed through the 30 kDa filter. Following ultrafiltering with the 30 kDa membrane, the permeate was collected and further concentrated using a 1 kDa membrane cartridge to remove low-MW compounds including sugars and salts. The protein concentration of the resulting 1 kDa retentate increased to 10.4 mg/mL, a 3-fold increase from the starting 30 kDa permeate. As expected, the protein concentration of the 1 kDa permeate was low at 2.1 mg/mL. The starting supernatant had a lunasin concentration of 18.2 ± 1.5 µg lunasin/mg protein, which was not statistically different (p > 0.05) from the lunasin concentration of the 30 kDa retentate (24.1 ± 2.8 µg lunasin/mg protein). On the other hand, the lunasin concentration of the 30 kDa permeate increased to 53.0 ± 11.0 µg lunasin/mg protein. The lunasin concentration of the 1 kDa retentate was similar to the 30 kDa permeate at 58.2 ± 11.4 µg lunasin/mg protein. This observation was expected as the 30 kDa permeate was only concentrated to obtain the 1 kDa retentate.  6 and 7). The identities of lunasin in these fractions were further validated by the western blot profiles of the samples indicating positive reactivity towards lunasin rabbit polyclonal antibody (Figure 3b). Several reports have been done on the isolation and purification of lunasin from defatted soy flour. For instance, Seber et al. [33] used a combination of anion-exchange chromatography, reduction technique, ultrafiltration, and reverse-phase chromatography to obtain a >99% lunasin purity. While Park et al. [34] used a combination of ion-exchange chromatography and ultrafiltration techniques to obtain purified soy lunasin. Krishnan and Wang [35] reported a method to enrich lunasin based on the extraction of soybean flour with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors using calcium. This process yields 3.2 g of lunasin and protease inhibitors from 100 g of soybean flour. The present report is the first on the production of lunasin-enriched soy extract using a two-step ultrafiltration technique. The concentration of lunasin in our prepared LESE was 58.2 µ g lunasin/mg protein; compared to the concentrations in commercial lunasin-enriched products (concentrations ranging from 9.2 to 25.7 µ g lunasin/mg protein) this process enriched lunasin by 2.3 to 6.3-fold.  6 and 7). The identities of lunasin in these fractions were further validated by the western blot profiles of the samples indicating positive reactivity towards lunasin rabbit polyclonal antibody (Figure 3b). Several reports have been done on the isolation and purification of lunasin from defatted soy flour. For instance, Seber et al. [33] used a combination of anion-exchange chromatography, reduction technique, ultrafiltration, and reverse-phase chromatography to obtain a >99% lunasin purity. While Park et al. [34] used a combination of ion-exchange chromatography and ultrafiltration techniques to obtain purified soy lunasin. Krishnan and Wang [35] reported a method to enrich lunasin based on the extraction of soybean flour with 30% ethanol followed by preferential precipitation of lunasin and protease inhibitors using calcium. This process yields 3.2 g of lunasin and protease inhibitors from 100 g of soybean flour. The present report is the first on the production of lunasin-enriched soy extract using a two-step ultrafiltration technique. The concentration of lunasin in our prepared LESE was 58.2 µg lunasin/mg protein; compared to the concentrations in commercial lunasin-enriched products (concentrations ranging from 9.2 to 25.7 µg lunasin/mg protein) this process enriched lunasin by 2.3 to 6.3-fold.   Results indicate that the antioxidant capacity and the inhibition potential of HAT may be partially attributed to lunasin present in the different soy products. However, phytochemicals such as saponins, isoflavones, among others could also be present and exert an effect on the antioxidant capacity. Previous studies have shown the antioxidant potential of lunasin in different in vitro models including RAW 264.7 macrophages, HepG2 cells, and Caco-2 cells [6,15,29]. On the other hand, studies on the chemopreventive property of lunasin have focused on its ability to alter the histone acetylation/deacetylation process. Lunasin in vitro is an inhibitor of H4 acetylation by p300/cAMP response element-binding protein-associated factor depending on the position of lysine-acetylated [36].  Results indicate that the antioxidant capacity and the inhibition potential of HAT may be partially attributed to lunasin present in the different soy products. However, phytochemicals such as saponins, isoflavones, among others could also be present and exert an effect on the antioxidant capacity. Previous studies have shown the antioxidant potential of lunasin in different in vitro models including RAW 264.7 macrophages, HepG2 cells, and Caco-2 cells [6,15,29]. On the other hand, studies on the chemopreventive property of lunasin have focused on its ability to alter the histone acetylation/deacetylation process. Lunasin in vitro is an inhibitor of H4 acetylation by p300/cAMP response element-binding protein-associated factor depending on the position of lysine-acetylated [36]. Nutrients 2021, 13, x FOR PEER REVIEW 10 of 14   Figure 6a shows cell viability of B16-F10 and A-375 after LE treatment. Results showed that lunasin is effective in a concentration-dependent manner. Lunasin had an IC50 = 1.84 ± 0.06 mg/mL (equivalent to 330 μM) after 24 h on B16-F10 and IC50 = 2.03 ± 0.05 mg/mL (equivalent to 370 μM) on A-375. Shidal et al. [37], reported lunasin to be effective in A-375 cells after 36 h treatment with 100 μM lunasin; however, 50% cell inhibition was not achieved in this study. Lunasin was showed to be more effective in decreasing cell    Figure 6a shows cell viability of B16-F10 and A-375 after LE treatment. Results showed that lunasin is effective in a concentration-dependent manner. Lunasin had an IC50 = 1.84 ± 0.06 mg/mL (equivalent to 330 μM) after 24 h on B16-F10 and IC50 = 2.03 ± 0.05 mg/mL (equivalent to 370 μM) on A-375. Shidal et al. [37], reported lunasin to be effective in A-375 cells after 36 h treatment with 100 μM lunasin; however, 50% cell inhibition was not achieved in this study. Lunasin was showed to be more effective in decreasing cell Samples were used at a concentration of 100 µg protein/mL; materials included: Soy Sentials ® , Slimplicity ® , Now ® dietary supplement, Provantage ® , Lunarich X™, GlucAffect ® , Now ® kids-vanilla and Now ® kids-chocolate (n = 3). Gallic acid (0.1 mg/mL) had an ORAC value of 220.3 ± 2.7 mmol TE/g, used for comparison purposes. Figure 6a shows cell viability of B16-F10 and A-375 after LE treatment. Results showed that lunasin is effective in a concentration-dependent manner. Lunasin had an IC 50 = 1.84 ± 0.06 mg/mL (equivalent to 330 µM) after 24 h on B16-F10 and IC 50 = 2.03 ± 0.05 mg/mL (equivalent to 370 µM) on A-375. Shidal et al. [37], reported lunasin to be effective in A-375 cells after 36 h treatment with 100 µM lunasin; however, 50% cell inhibition was not achieved in this study. Lunasin was showed to be more effective in decreasing cell viability in A-375 melanoma cells after 24 h than oleuropein a bioactive compound from olive leaves, which needed 800 µM to decrease~50% after 24 h [38].

Protein Kinase B (Akt) Pathway Expression
The most common mutation in melanoma is located on the BRAF gene [42,43] which activates sequentially mitogen-activated protein kinase (MEK 1/2) and extracellular signal-regulated kinase (ERK1/2) leading to cell proliferation [42]. Previous studies reported that lunasin decreased phosphorylated (p-)ERK [19,44], we hypothesized that the cell growth inhibition could be due to an ERK inhibition. After 24 h treatment with IC50 in B16-F10 and A-375, the expression of p-AKT pathway ( Figure 6) showed significant ERK inhibition (p < 0.05) for B16-F10. The inhibition of PRAS40 produced inhibition in mTOR, ribosomal protein S6 (RPS6), ribosomal protein S6 kinase (P70S6k), and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) interfering with cell growth and metabolism [45]. Lv et al. [45] reported that melanoma with high AKT activity also showed an increase in phosphorylated PRAS40.

Protein Kinase B (Akt) Pathway Expression
The most common mutation in melanoma is located on the BRAF gene [42,43] which activates sequentially mitogen-activated protein kinase (MEK 1/2) and extracellular signalregulated kinase (ERK1/2) leading to cell proliferation [42]. Previous studies reported that lunasin decreased phosphorylated (p-)ERK [19,44], we hypothesized that the cell growth inhibition could be due to an ERK inhibition. After 24 h treatment with IC 50 in B16-F10 and A-375, the expression of p-AKT pathway ( Figure 6) showed significant ERK inhibition (p < 0.05) for B16-F10. The inhibition of PRAS40 produced inhibition in mTOR, ribosomal protein S6 (RPS6), ribosomal protein S6 kinase (P70S6k), and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) interfering with cell growth and metabolism [45]. Lv et al. [45] reported that melanoma with high AKT activity also showed an increase in phosphorylated PRAS40.
Since p-glycogen synthase kinase 3 (GSK3) was significant in B16-F10 (p < 0.005) and A375 (p < 0.05) (Figure 6), our data suggested that lunasin promotes GSK3β inhibition through Ser9 phosphorylation. GSK3 functions in different cell processes including glycogen metabolism, proliferation, differentiation, motility, and survival. It could act as a tumor promoter or tumor suppressor [46][47][48]. GSK3β requires phosphorylation at Tyr216 for full activity, whereas, phosphorylation in Ser9 will result in an inactivation, which is the most important regulatory mechanism [46,48]. There is evidence that GSK3β inhibition limits motility and invasion of melanoma cells, which could lead to limit the metastatic behavior via N-cadherin signaling inhibition [49]. In an in vivo study where mice were fed with a soy powder mixture containing 52.6% of protein, 30.1% carbohydrates, and 5% lipids, inhibition on N-cadherin was shown [50]. Further studies to investigate any relationship between lunasin and N-cadherin signaling are needed.
As shown in Figure 6 and Table 2, AKT and RPS6 expressions were higher than the control for A-375 in 37% and 24%, respectively. A non-significant 24% overexpression in BAD compared with the control, however, was shown in the same cell line, which suggests that AKT and RPS6 overexpression, in this case, could not affect the cell growth.

Conclusions
The concentration of lunasin in different commercial lunasin-based dietary supplements was comparable to commercial soymilk and soy-based infant formulas. A simple two-step pilot-plant ultrafiltration process to generate lunasin-enriched soy extract was developed. This process is an alternative for enriching lunasin content in soybean products. Moreover, a significant positive correlation between ORAC and lunasin concentrations and a significant negative correlation between HAT activities and lunasin concentrations were found. Lunasin concentration in lunasin-based commercial dietary supplements and lunasin-enriched soy extract could increase the potential health benefits associated with soy consumption. Lunasin cytotoxicity in both melanoma cells was dose-dependent reaching a half-maximum inhibitory concentration at a concentration equivalent to 330 µM for B16-F10 and 370 µM for A375.