Soybean Lunasin Research Articles

Encapsulation of soybean lunasin and amaranth unsaponifiable matter in liposomes induces cell cycle arrest in an allograft melanoma mouse model

Melanoma is the most aggressive type of skin cancer and can metastasize during primary tumor formation. This research aimed to determine the relationship between the prevention of melanoma development in a mouse model treated with liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) and cell cycle arrest. Tumors excised from C57BL/6 mice treated topically or subcutaneously with UM + LunLip were subjected to immunohistochemistry. Markers related to cell cycle inhibition (p16, p21, p27, and p53) and markers involved in cell cycle progression (cyclin-dependent kinase, CDK6, and cyclin D1) were assessed. The results showed that UM + LunLip had antitumor activity in C57BL/6 mice treated either topically or subcutaneously by p16, p21, p27, and p53 overexpression (up to 572-, 134-, 30-, and 57-fold change, FC, respectively) in the tumors of mice treated with 30 mg UM + LunLip/kg body weight compared with the tumor-bearing untreated control. However, CDK6 and cyclin D1 expression was not inhibited (up to 1.37 FC and 2.09 FC, respectively), which is a typical behavior of cyclin D in melanoma. Therefore, melanoma tumor development was prevented by the overexpression of cell cycle inhibitors p16, p21, p27, and p53 due to UM + LunLip treatments. Since the topical application was effective, less invasive, and more practical for the user, this application will be recommended for future steps in in vivo studies.

Liposomes Loaded with Amaranth Unsaponifiable Matter and Soybean Lunasin Prevented Melanoma Tumor Development Overexpressing Caspase-3 in an In Vivo Model.

The objective of this study was to assess the effectiveness of liposomes loaded with soybean lunasin and amaranth unsaponifiable matter (UM + LunLip) as a source of squalene in the prevention of melanoma skin cancer in an allograft mice model. Tumors were induced by transplanting melanoma B16-F10 cells into the mice. The most effective treatments were those including UM + LunLip, with no difference between the lunasin concentrations (15 or 30 mg/kg body weight); however, these treatments were statistically different from the tumor-bearing untreated control (G3) (p < 0.05). The groups treated with topical application showed significant inhibition (68%, p < 0.05) compared to G3. The groups treated with subcutaneous injections showed significant inhibition (up to 99%, p < 0.05) in G3. During tumor development, UM + LunLip treatments under-expressed Ki-67 (0.2-fold compared to G3), glycogen synthase kinase-3β (0.1-fold compared to G3), and overexpressed caspase-3 (30-fold compared to G3). In addition, larger tumors showed larger necrotic areas (38% with respect to the total tumor) (p < 0.0001). In conclusion, the UM + LunLip treatment was effective when applied either subcutaneously or topically in the melanoma tumor-developing groups, as it slowed down cell proliferation and activated apoptosis.

Liposomes Containing Amaranth Unsaponifiable Matter and Soybean Lunasin Suppress ROS Production in Fibroblasts and Reduced Interleukin Production in Macrophages.

Inflammation is a normal response in defense to agents that may cause damage to the human body. When inflammation becomes chronic, reactive oxygen species (ROS) are produced; which could lead to diseases such as cancer. The aim was to assess liposomes’ antioxidant and anti-inflammatory capacity loaded with amaranth unsaponifiable matter and soybean lunasin (UM + LunLip) in an in vitro model using fibroblasts and macrophages. To evaluate ROS production, fibroblasts CHON-002 ABAP were added to promote ROS production; and the cells were treated with UM + LunLip. For inflammation markers production, lipopolysaccharides (LPS)-stimulated RAW 264.7 and peritoneal macrophages were treated with empty liposomes (EmLip), liposomes loaded with unsaponifiable matter (UMLip), liposomes loaded with lunasin (LunLip), and UM + LunLip. ROS production was significantly decreased by 77% (p < 0.05) when fibroblasts were treated with UM + LunLip at 2 mg lunasin/mL compared with the control treated with ABAP. Treatment with UMLip was the most effective in reducing tumor necrosis factor-α (71–90%) and interleukin-6 (43–55%, p < 0.001). Both liposomes containing unsaponifiable matter (UMLip and UM + LunLip) were more effective than EmLip or LunLip. In conclusion, amaranth unsaponifiable matter-loaded liposomes are effective in decreasing pro-inflammatory cytokine production.

Liposomes Loaded With Soybean Lunasin and Amaranth Unsaponifiable Matter Promoted Apoptosis Through Caspase 3 and Cell Proliferation Arrest in an In Vivo Melanoma Model

ObjectivesThe objective was to assess the mechanism of action of liposomes loaded with amaranth unsaponifiable matter and lunasin (LpLnUM) in melanoma tumors developed in C57BL/6 mice. MethodsTumors were developed in C57BL/6 using the melanoma cell line B16-F10 and treated every other day for 22 days using LpLnUM with 15 (LpLnUM15) or 30 (LpLnUM30) mg lunasin/kg body weight (BW) via either topically (T) or subcutaneously (S) (treated groups, TG), or lunasin solution at 30 mg/kg BW. Tumors were excised, fixed, and embedded in paraffin, cut with a microtome (5 μm thick), and fixed in microscope slides. For immunohistochemistry, glycogen synthase kinase (GSK)-3β, caspase 3 (C3), and Ki-67 antibodies were used, with horseradish peroxidase and 3,3’-diaminobenzidine as a substrate to reveal protein expression. Hematoxylin and eosin assay assessed tissue structure. Images were captured with a NanoZoomer and analyzed at a 20x magnification. ResultsCompared with the tumor-untreated control (UC), T and S applied liposomes inhibited the tumor in 61 and 71%, respectively. No difference between lunasin concentrations (p < 0.05). The smaller the tumor volume, the higher C3 expression (r = –0.819, p < 0.05). Tumors showed a C3 overexpression in those groups S treated with LpLnUM15 (1.41-fold, p < 0.01) and LpLnUM30 (1.53-fold, p < 0.01) compared with the UC. In contrast, lunasin solution (without liposomes) T applied at 30 mg/kg BW had no difference with the UC, but it was different (p < 0.05) when compared with the groups treated with only S applied liposomes. GSK-3β suggested that even though the expression decreased in the TG there was no difference among controls and treatments. The proliferation marker Ki-67 showed an under expression of the marker between the TG (p < 0.001) and the UC. The Ki-67 expression in mice T treated with LpLnUM30 and LpLnUM15 was inhibited 89 and 76%, respectively. In those mice with S application, the Ki-67 inhibition was 81 and 93% when treated with LpLnUM30 and LpLnUM15, respectively. These last results suggest an arrest in the cell cycle. ConclusionsLpLnUM15 and LpLnUM30 prevented melanoma tumor development by promoting cell apoptosis via C3 expression and cell cycle arrest regardless of lunasin concentration and application method in mice. Funding SourcesUSDA National Institute of Food and Agriculture, CONACyT fellowship.

Liposomes Loaded with Unsaponifiable Matter from Amaranthus hypochondriacus as a Source of Squalene and Carrying Soybean Lunasin Inhibited Melanoma Cells.

Amaranthus hypochondriacus is a source of molecules with reported health benefits such as antioxidant activity and cancer prevention. The objective of this research was to optimize the conditions for preparing a liposome formulation using amaranth unsaponifiable matter as a source of squalene in order to minimize the particle size and to maximize the encapsulation efficiency of liposomes for carrying and delivering soybean lunasin into melanoma cell lines. Amaranth oil was extracted using supercritical dioxide carbon extraction (55.2 MPa pressure, 80 °C temperature, solvent (CO2)-to-feed (oil) ratio of 20). The extracted oil from amaranth was used to obtain the unsaponifiable enriched content of squalene, which was incorporated into liposomes. A Box–Behnken response surface methodology design was used to optimize the liposome formulation containing the unsaponifiable matter, once liposomes were optimized. Soybean lunasin was loaded into the liposomes and tested on A-375 and B16-F10 melanoma cells. The squalene concentration in the extracted oil was 36.64 ± 0.64 g/ 100 g of oil. The particle size in liposomes was between 115.8 and 163.1 nm; the squalene encapsulation efficiency ranged from 33.14% to 76.08%. The optimized liposome formulation contained 15.27 mg of phospholipids and 1.1 mg of unsaponifiable matter. Cell viability was affected by the liposome formulation with a half-maximum inhibitory concentration (IC50) equivalent to 225 μM in B16-F10 and 215 μM in A-375. The liposomes formulated with lunasin achieved 82.14 ± 3.34% lunasin encapsulation efficiency and improved efficacy by decreasing lunasin IC50 by 31.81% in B16-F10 and by 41.89% in A-375 compared with unencapsulated lunasin.

The Effect of Soybean Lunasin on Cardiometabolic Risk Factors: A Randomized Clinical Trial

Lunasin, a peptide isolated from soybeans, has been shown to exert antioxidant, anticarcinogenic, and hypocholesterolemic effects in animal and in vitro models. In a triple-blind, placebo-controlled crossover study, 31 participants (female: 19, male: 12, mean age 61 ± 9.9 years) were randomized to an 8-week treatment of lunasin-enriched soy extract (335mg/d) or placebo. A 3-week washout period was utilized between treatments. Serum lipids, glucose, and insulin, as well as blood pressure and anthropometrics, were measured at baseline, week 7, and week 8 of each treatment period. There were nonsignificant reductions in cardiometabolic risk factors with treatment: total cholesterol −0.1mmol/L, 95% CI [−0.28, 0.03]; LDL cholesterol −0.07mmol/L, 95% CI [−0.2, 0.06]; triglyceride 0% mmol/L, 95% CI (−10%, 11%]; fasting serum glucose −2% mmol/L, 95% CI [−4%, 1%]; BMI −0.05 kg/m2, 95% CI [−0.17, 0.07] and waist circumference −0.63cm, 95% CI [−1.8, 0.53]. Supplementation with lunasin-enriched soy extract for 8 weeks did not result in significant changes in serum lipids, glucose, insulin resistance, blood pressure, BMI, or waist circumference. Future studies should focus on a higher dosage, larger sample size, and/or longer treatment to determine the independent role of lunasin, if any, in the effect of soy on cardiometabolic risk factors.

Revealing the regional distribution of soybean lunasin content in China and the effects of climate factors by sampling extensively.

Lunasin is a novel therapeutic peptide that was initially isolated from soybean. In this study, we quantified the variations in lunasin content in a total of 413 soybean samples that were collected from four major regions in China and harvested in 2014 and 2015 to reveal the regional distribution of soybean lunasin content in China and the effects of climate factors. The results showed that the cultivar Changmidou 30 collected from Jilin province and harvested in 2015 had the highest lunasin content (3.25 g kg-1 dry seeds). The data from both 2014 and 2015 showed that the lunasin content in soybean collected from northern China was significantly higher (P<0.05) than that from south China. There was a positive correlation (P<0.01) between lunasin content and hours of sunshine (HS) as well as diurnal temperature range (DTR); however, there was a negative correlation (P<0.01) between lunasin content and rainfall (RF). In addition, combined analysis of data from 2014 and 2015 demonstrated that DTR was the dominant factor that affected lunasin content with a direct path-coefficient of 0.301. These results were anticipated to contribute to guiding the cultivation of soybean with high lunasin content. © 2018 Society of Chemical Industry.

Immunological Investigation for the Presence of Lunasin, a Chemopreventive Soybean Peptide, in the Seeds of Diverse Plants.

Lunasin, a 44 amino acid soybean bioactive peptide, exhibits anticancer and anti-inflammatory properties. All soybean varieties that have been examined contain lunasin. It has also been reported in a few other plant species including amaranth, black nightshade, wheat, barley, rye, and triticale. Interestingly, detailed searches of transcriptome and DNA sequence databases of cereals failed to identify lunasin-coding sequences, raising questions about the authenticity of lunasin in cereals. To clarify the presence or absence of lunasin in cereals and other plant species, an immunological investigation was conducted utilizing polyclonal antibodies raised against the first 20 amino acid N-terminal peptide (SKWQHQQDSCRKQLQGVNLT) and a 15 amino acid C-terminal peptide (CEKHIMEKIQGRGDD) of lunasin. Protein blot analyses revealed the presence of proteins from several plants that reacted against the lunasin N-terminal peptide antibodies. However, the same proteins failed to react against the lunasin C-terminal peptide antibodies. These results demonstrate that peptides identical to soybean lunasin are absent in seeds of diverse plants examined in this study.

Lunasin detection in coloured wheat genotype

Lunasin is a biologically active protein, composed of 43 amino acid residues. There has been proven many health-promoting effects of lunasin peptide. The most important health benefits include: anti-hypertension, antioxidant activity, cancer prevention or therapy. It was also demonstrated anti-inflammation, hypocholesterolemic activity, anti-obesity and immunomodulation. The focus of our research is to summarize the discovery, characterization and biological activities of lunasin, which will provide a reference for the future development and utilization of lunasin, and a basis for exploring the underlying mechanisms of these health-beneficial functions. Lunasin was first isolated in 1987 at Niigata University School of Medicine in Japan, during the screening of protease inhibitors from soybean seeds. It was subsequently found in other beans, grains and herbal plants, including wheat, barley, rye, triticale. Concentration of lunasin is ranging from 0.013 to 70.5 mg protein lunasin/g of protein. Big step forward in the understanding of the lunasine operating mechanism in the fight against cancer has arisen after study on cloning of the soybean lunasin gene and subsequent transfection into mammalian cells which led to the discovery that the lunasin gene can disrupt mitosis and induce chromosome breakage, ultimately leading to cell apoptosis. The main goal of our work was to evaluate collection of wheat with unusual grain colour for presence of lunasin gene. DNA was extracted by commercial kit and lunasin gene was detected by PCR reaction. Our results showed presence of lunasin gene detected by 3 combinations of 2 sets of primer pair and indicated lunasin peptide presence in cereal grains. These findings are necessary to confirmed by proteome analysis.

The protective role of the Bowman-Birk protease inhibitor in soybean lunasin digestion: the effect of released peptides on colon cancer growth.

Lunasin is a naturally-occurring peptide demonstrating chemopreventive, antioxidant and anti-inflammatory properties. To exhibit these activities, orally ingested lunasin needs to survive proteolytic attack of digestive enzymes to reach target tissues in active form/s. Preliminary studies suggested the protective role of protease inhibitors, such as the Bowman-Birk inhibitor and Kunitz-trypsin inhibitor, against lunasin's digestion by both pepsin and pancreatin. This work describes in depth the behaviour of lunasin under conditions simulating the transit through the gastrointestinal tract in the absence or presence of soybean Bowman-Birk isoinhibitor 1 (IBB1) in both active and inactive states. By liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS), the remaining lunasin at the end of gastric and gastro-duodenal phases was quantified. Protection against the action of pepsin was independent of the amount of IBB1 present in the analyzed samples, whereas an IBB1 dose-dependent protective effect against trypsin and chymotrypsin was observed. Peptides released from lunasin and inactive IBB1 were identified by MS/MS. The remaining lunasin and IBB1 as well as their derived peptides could be responsible for the anti-proliferative activity against colon cancer cells observed for the digests obtained at the end of simulated gastrointestinal digestion.

Structural property of soybean lunasin and development of a method to quantify lunasin in plasma using an optimized immunoassay protocol

Lunasin is a 43-amino acid naturally occurring chemopreventive peptide with demonstrated anti-cancer and anti-inflammatory properties. The objectives of this study were to determine the effect of temperature on the secondary structure of lunasin, to develop a method of isolating lunasin from human plasma using an ion-exchange microspin column and to quantify the amount of lunasin using an optimized enzyme-linked immunosorbent assay. Lunasin was purified using a combination of ion-exchange chromatography, ultrafiltration and gel filtration chromatography. Circular dichroism showed that increased in temperature from 25 to 100°C resulted in changes on the secondary structure of lunasin and its capability to interact with rabbit polyclonal antibody. Enzyme linked immunosorbent assay showed that lunasin rabbit polyclonal antibody has a titer of 250 and a specific activity of 0.05mL/μg. A linear response was detected between 16 to 48ng lunasin per mL (y=0.03x−0.38, R2=0.96). The use of diethylaminoethyl microspin column to isolate spiked lunasin in human plasma showed that most lunasin (37.8–46.5%) bound to the column eluted with Tris–HCl buffer, pH 7.5 with a yield up to 76.6%. In conclusion, lunasin can be isolated from human plasma by a simple DEAE microspin column technique and can be quantified using a validated and optimized immunoassay procedure. This method can be used directly to quantify lunasin from plasma in different human and animal studies aiming to determine its bioavailability.

Amaranth lunasin-like peptide internalizes into the cell nucleus and inhibits chemical carcinogen-induced transformation of NIH-3T3 cells

Because an unbalanced diet is an important risk factor for several illnesses, interest has increased in finding novel health-promoting foods. Amaranth produces seeds that not only have substantial nutritional properties but that also contain phytochemical compounds as rutin and nicotiflorin and peptides with antihypertensive and anticarcinogenic activities. We report that a cancer-preventive peptide in amaranth has activities similar to those of soybean lunasin. The amaranth lunasin-like peptide, however, requires less time than the soybean lunasin to internalize into the nucleus of NIH-3T3 cells, and inhibits histone acetylation (H 3 and H 4 in a 70 and 77%, respectively). The amaranth lunasin-like peptide inhibited the transformation of NIH-3T3 cells to cancerous foci. The open reading frame (ORF) of amaranth lunasin corresponds to a bifunctional inhibitor/lipid-transfer protein (LTP). LTPs are a family of proteins that in plants are implicated in different functions, albeit all linked to developmental processes and biotic and abiotic stress resistance. Our results open new intriguing questions about the function of lunasin in plants and support that amaranth is a food alternative containing natural peptides with health-promoting benefits.

Bioactive Peptides in Amaranth (Amaranthus hypochondriacus) Seed

Amaranth seeds are rich in protein with a high nutritional value, but little is known about their bioactive compounds that could benefit health. The objectives of this research were to investigate the presence, characterization, and the anticarcinogenic properties of the peptide lunasin in amaranth seeds. Furthermore, to predict and identify other peptides in amaranth seed with potential biological activities. ELISA showed an average concentration of 11.1 microg lunasin equivalent/g total extracted protein in four genotypes of mature amaranth seeds. Glutelin fraction had the highest lunasin concentration (3.0 microg/g). Lunasin was also identified in albumin, prolamin and globulin amaranth protein fractions and even in popped amaranth seeds. Western blot analysis revealed a band at 18.5 kDa, and MALDI-TOF analysis showed that this peptide matched more than 60% of the soybean lunasin peptide sequence. Glutelin extracts digested with trypsin, showed the induction of apoptosis against HeLa cells. Prediction of other bioactive peptides in amaranth globulins and glutelins were mainly antihypertensive. This is the first study that reports the presence of a lunasin-like peptide and other potentially bioactive peptides in amaranth protein fractions.

Characterization of lunasin isolated from soybean.

Lunasin is a novel and promising chemopreventive peptide from soybean. We have shown previously that lunasin suppresses transformation of mammalian cells caused by chemical carcinogens and inhibits skin carcinogenesis in mice when applied topically. Although the lunasin gene was cloned from soybean, all experiments carried out so far in our lab have used synthetic lunasin and therefore there is no detailed description of natural lunasin isolated from soybean. We report here the first characterization of soybean lunasin that includes definitive identification by mass peptide mapping, partial purification, and measurement of bioactivities of the various purified fractions and protein expression in the developing seed. The identity of lunasin in the seed extracts was established by Western blot analysis and mass spectrometric peptide mapping. All lunasin fractions partially purified by anion exchange and immunoaffinity column chromatography suppress colony formation induced by the ras-oncogene and inhibit core H3-histone acetylation. During seed development, lunasin peptide appears 5 weeks after flowering and persists in the mature seed. Western blot analysis of different soybean varieties and commercially available soy proteins shows the presence of the peptide in varying amounts. These results demonstrate the feasibility of producing large quantities of natural lunasin from soybean for animal and human studies.