Corn peptide having Anti-tumor effect, and preparation method therefor and use thereof

Abstract

Provided are a corn peptide having an anti-tumor effect, and a preparation method therefor and a use thereof. The corn peptide can obviously eliminate reactive oxygen species in tumor cells, increase the concentration of free calcium ions in the cytoplasm of the tumor cells and reduce the mitochondrial membrane potential of the tumor cells, and also can enhance the expression levels of mRNA and proteins of an IP3R and calreticulin, and further affect the calcium homeostasis of the tumor cells. In addition, the corn peptide can up-regulate the expression of tumor suppressor genes PTEN, P21 and P27, down-regulate the expression of a cell proliferation marker PCNA and a tumor-promoting gene Akt, and mediate an anti-tumor effect by means of PTEN and Akt cell signaling pathways.

Description

Corn peptide with anti-tumor effect and preparation method and application thereof

[0001] The present application claims priority to the Chinese patent application No. 202411001359.0, filed on July 25, 2024, and entitled "Corn peptide with anti-tumor effect and preparation method and application thereof", the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present application relates to biotechnology, in particular to a corn peptide with anti-tumor effect and a preparation method and application thereof. BACKGROUND

[0003] Tumor is a new biological formed by abnormal proliferation of a cell in local tissue under the action of various carcinogenic factors at the genetic level, which loses normal regulation of its growth. Generally, tumors are divided into two categories: benign and malignant. Benign tumors are generally referred to as "tumor", and malignant tumors from epithelial tissue are referred to as "carcinoma", and those from mesenchymal tissue are referred to as "sarcoma". On February 2, 2024, the International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) released the Global cancer burden growing, amid mounting need for services, which once again emphasized the growing global cancer burden, which deserves attention worldwide. In summary, the number of new cancer cases worldwide in 2022 reached 20 million, and the number of deaths was 9.7 million; it is estimated that the number of survivors 5 years after diagnosis is 53.5 million, and about 1 / 5 of people will suffer from cancer in their lifetime, and about 1 / 9 of men and 1 / 12 of women will die of cancer. Therefore, how to inhibit and treat tumors needs to be solved.

[0004] SUMMARY

[0005] The present application provides a corn peptide with anti-tumor effect, which can affect the calcium homeostasis of tumor cells and mediate the anti-tumor cell effect through the PTEN and Akt cell signaling pathways.

[0006] The present application provides a preparation method of the corn peptide, which uses corn protein as raw material, and obtains the corn peptide through enzymatic treatment and separation and purification treatment in sequence, which is simple and fast in operation and convenient for large-scale production.

[0007] The present application also provides an application of the corn peptide in preparing anti-tumor related products. The corn peptide of the present application can affect the calcium homeostasis of tumor cells and mediate the anti-tumor cell effect through the PTEN and Akt cell signaling pathways, and therefore can be used in preparing anti-tumor related products.

[0008] The application provides corn peptides with anti-tumor effect, wherein the corn peptides at least comprise peptide segments AP, IP and VAYPE.

[0009] Based on the mass of the corn peptides, the content of the peptide segment AP is greater than or equal to 8.3 mg / 100 g, the content of the peptide segment IP is greater than or equal to 33.95 mg / 100 g, and the content of the peptide segment VAYPE is greater than or equal to 4.26 mg / 100 g.

[0010] The corn peptides as described above, wherein the content of protein in the corn peptides is greater than or equal to 84.47 g / 100 g, the content of amino acids is greater than or equal to 80.56 g / 100 g, and the mass content of peptides with a molecular weight less than 1000 u is greater than or equal to 97%.

[0011] The corn peptides as described above, wherein the corn peptides are obtained by using corn protein as raw material, and sequentially performing enzymolysis treatment and separation and purification treatment.

[0012] The enzymolysis treatment comprises using alkaline protease and bromelain for enzymolysis.

[0013] The application provides a preparation method of the corn peptides, comprising the following steps:

[0014] 1) performing first enzymolysis on corn protein by using alkaline protease to obtain a first enzymolysis solution, and performing second enzymolysis on the first enzymolysis solution by using bromelain to obtain a second enzymolysis solution;

[0015] 2) performing separation and purification treatment on the second enzymolysis solution to obtain corn peptides.

[0016] The preparation method as described above, wherein based on per gram of corn protein, the enzyme activity of the alkaline protease is 2.4 AU, and the enzyme activity of the bromelain is 50000-100000 U.

[0017] The preparation method as described above, wherein the temperature of the first enzymolysis is 50-65 ℃, the time is 20-45 min, and the pH value is 8.5; and / or,

[0018] The temperature of the second enzymolysis is 40-60 ℃, the time is 110-130 min, and the pH value is 7.

[0019] The preparation method as described above, wherein the separation and purification treatment comprises sequentially performing filtration treatment, activated carbon adsorption treatment, cation exchange resin treatment and anion exchange resin treatment on the second enzymolysis solution.

[0020] The preparation method as described above, wherein the filtration treatment comprises: performing filtration treatment by using a filter membrane with a pore size of 200 nm.

[0021] The preparation method as described above, wherein the activated carbon adsorption treatment comprises: using activated carbon with a particle size of ≤0.075mm, and performing activated carbon adsorption treatment at a temperature of 40-60℃ for 30-40min.

[0022] The application also provides application of the corn peptide in preparation of a product related to anti-tumor cells.

[0023] The application provides a corn peptide with an anti-tumor effect, which can obviously remove active oxygen in tumor cells, increase the concentration of free calcium ions in the cytoplasm of tumor cells, reduce the mitochondrial membrane potential of tumor cells, and also can enhance the expression amount of mRNA and protein of IP3R and calnexin, further affect the calcium homeostasis of tumor cells. Meanwhile, the corn peptide can also up-regulate the expression of cancer-suppressing genes PTEN, P21 and P27, down-regulate the expression of cell proliferation markers PCNA and cancer-promoting genes Akt, and mediate the anti-tumor effect through the PTEN and Akt cell signaling pathways. BRIEF DESCRIPTION OF DRAWINGS

[0024] Fig. 1 is a product ion mass spectrum of the peptide segment AP in Example 2;

[0025] Fig. 2 is a product ion mass spectrum of the peptide segment IP in Example 2;

[0026] Fig. 3 is a product ion mass spectrum of the peptide segment VAYPE in Example 2;

[0027] Fig. 4 is a standard curve of the peptide segment AP in Example 2;

[0028] Fig. 5 is a standard curve of the peptide segment IP in Example 2;

[0029] Fig. 6 is a standard curve of the peptide segment VAYPE in Example 2;

[0030] Fig. 7 is a quantitative chromatogram of the target peptide segment of the peptide segment AP in Example 2;

[0031] Fig. 8 is a quantitative chromatogram of the target peptide segment of the peptide segment IP in Example 2;

[0032] Fig. 9 is a quantitative chromatogram of the target peptide segment of the peptide segment VAYPE in Example 2;

[0033] Fig. 10 is a gel chromatogram of the molecular weight distribution of the corn peptide in Example 2;

[0034] Fig. 11 is a cell viability detection result graph in Example 3;

[0035] Fig. 12 is a graph of the intracellular active oxygen level detection result in Example 3;

[0036] Fig. 13 is a graph of the intracellular Ca 2+Concentration and mitochondrial membrane potential detection results chart;

[0037] Figure 14 is a column chart of the protein expression of IP3R, Calreticulin, PTEN, P21, P27 in cells in Example 3;

[0038] Figure 15 is a column chart of the protein expression of PCNA, Akt1 and Akt2 in cells in Example 3;

[0039] Figure 16 is a protein immunoblotting chart of IP3R, Calreticulin, PTEN, P21, P27, Akt1 and Akt2 in cells in Example 3. DETAILED DESCRIPTION

[0040] In order for those skilled in the art to better understand the scheme of the present application, the present application is further described in detail below. The following specific embodiments are only used to describe the principles and characteristics of the present application, and the examples are used to explain the present application, but not to limit the scope of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0041] The first aspect of the present application provides a corn peptide (Cp) with anti-tumor effect, which at least includes dipeptide segments (Ala-Pro, AP) composed of alanine and proline, dipeptide segments (Ile-Pro, IP) composed of isoleucine and proline, and polypeptide segments (Val-Ala-Tyr-Pro-Glu, VAYPE) composed of valine, alanine, tyrosine, proline and glutamic acid; based on the mass of corn peptide (dry basis), the content of peptide segment AP is ≥8.3 mg / 100 g, the content of peptide segment IP is ≥33.95 mg / 100 g, and the content of peptide segment VAYPE is ≥4.26 mg / 100 g.

[0042] The present application regulates the functional peptide segments in the composition of corn peptide and their contents, so that it shows good efficacy in anti-tumor. The corn peptide of the present application has anti-tumor effect, can obviously remove active oxygen in tumor cells, increase the concentration of free calcium ions in the cytoplasm of tumor cells, reduce the mitochondrial membrane potential of tumor cells, and can also enhance the mRNA and protein expression of IP3R and Calreticulin, further affecting the calcium homeostasis of tumor cells. At the same time, the corn peptide can also up-regulate the expression of tumor suppressor genes PTEN, P21 and P27, down-regulate the expression of cell proliferation marker PCNA and oncogene Akt, and mediate anti-tumor effect through PTEN and Akt cell signaling pathways.

[0043] Further, the content of protein in the corn peptide (dry basis) is ≥ 84.47 g / 100 g, the content of amino acid is ≥ 80.56 g / 100 g, and the content of free amino acid is ≤ 3.74 g / 100 g. In addition, the corn peptide has the characteristics of small average molecular weight and easy absorption. Specifically, the mass content of peptides with a molecular weight less than 1000 u in the corn peptide is ≥ 97%.

[0044] The corn peptide of the present application is obtained by sequentially performing enzymatic treatment and separation and purification treatment on corn protein. The enzymatic treatment includes using alkaline protease and bromelain for enzymatic hydrolysis.

[0045] The present application does not limit the specific type of corn protein, as long as the content of protein in the corn protein (dry basis) is not less than 60%. It can be understood that the corn protein of the present application can be in solid or liquid form. In an embodiment of the present application, the corn protein used is corn gluten meal.

[0046] Corn gluten meal (CGM) is the largest by-product with the highest protein content in the process of wet corn starch production, which is rich in protein and corn flavonoids. However, corn gluten meal has many disadvantages such as poor water solubility, strong hydrophobicity, and difficulty in being digested by the human body, which limits the application of corn gluten meal, resulting in that most corn gluten meal is directly used as feed, causing great waste of food resources.

[0047] In order to further improve the utilization rate of corn gluten meal, corn gluten meal can be used as raw material for directional enzyme cutting and specific small peptide separation technology to obtain small molecular polypeptide material, also known as corn peptide. Corn peptide is different from corn gluten meal, and has the characteristics of direct absorption, strong solubility, strong stability, and high safety. At present, some studies have shown that corn peptide has various biological activities, such as anti-hypertension, immune enhancement, and anti-fatigue.

[0048] The present application uses alkaline protease and bromelain to perform enzymatic treatment on corn protein. The alkaline protease refers to an enzyme that can hydrolyze the peptide bond of protein under alkaline conditions, and its optimum pH range is 9-11. The bromelain refers to a sulfhydryl protease extracted from pineapple juice, pineapple peel, etc., which preferentially hydrolyzes the peptide chain on the carboxyl side of basic amino acids or aromatic amino acids, selectively hydrolyzes fibrous protein, and can decompose muscle fibers. Different proteins have different optimum pH ranges.

[0049] By regulating the enzymatic treatment and separation and purification treatment of corn protein, the corn peptide obtained finally can meet the preset functional peptide segment composition.

[0050] The second aspect of the present application provides a preparation method of the above corn peptide, comprising the following steps:

[0051] 1) using alkaline protease for the first enzymolysis of corn protein to obtain a first enzymolysis solution; using bromelain for the second enzymolysis of the first enzymolysis solution to obtain a second enzymolysis solution;

[0052] 2) performing separation and purification treatment on the second enzymolysis solution to obtain corn peptides.

[0053] The applicant has made a lot of research and exploration on how to make the enzymolysis product of corn protein contain the peptide segments AP, IP and VAYPE in the expected mass content. It is proved that the selection of enzyme preparation, enzyme treatment process and the corresponding separation and purification process have a key influence on the result. The applicant accidentally found that only by using alkaline protease and bromelain for successive enzymolysis and performing separation and purification treatment on the enzymolysis product, corn peptides containing the content of peptide segment AP ≥ 8.3 mg / 100 g, the content of peptide segment IP ≥ 33.95 mg / 100 g and the content of peptide segment VAYPE ≥ 4.26 mg / 100 g can be obtained.

[0054] In the scheme of the present application, if the corn protein is in a solid form, the corn protein can be mixed with water to obtain a corn protein solution. For example, when the corn protein is corn yellow powder, the corn yellow powder can be mixed with water according to a mass ratio of 1:(5-20), that is, 100 g of corn yellow powder is mixed with 0.5-2.0 L of water to prepare a corn yellow powder solution. The corn yellow powder solution has a certain flowability and a larger surface area than the corn yellow powder, which is beneficial to subsequent enzymolysis. If too little water is added, the flowability is poor, which is not conducive to the action of enzyme preparation and is easy to cause the enzymolysis efficiency to decrease; if too much water is added, the reaction volume is too large during the enzymolysis treatment, which increases the load of subsequent treatment (such as separation and purification treatment, etc.) and also increases the treatment cost accordingly. In addition, due to the poor water solubility of corn yellow powder, the corn yellow powder and water can be fully mixed by stirring, wherein the stirring speed is 200-300 rpm / min and the time is 30-50 min.

[0055] The enzymolysis treatment can be divided into two processes of first enzymolysis and second enzymolysis. First, alkaline protease is used for the first enzymolysis of the corn yellow powder solution to obtain a first enzymolysis solution, and then bromelain is used for the second enzymolysis of the first enzymolysis solution to obtain a second enzymolysis solution.

[0056] After the enzymolysis treatment, separation and purification treatment is performed on the second enzymolysis solution to further enrich the functional peptide segments. After the end of the separation and purification treatment, drying treatment can also be performed to obtain powdery corn peptides. Specifically, spray drying treatment can be performed, and the spray drying conditions are: inlet temperature 160℃ and outlet temperature 65℃.

[0057] By the preparation method of the corn peptide of the present application, not only the peptide segments AP, IP, VAYPE can be obtained, but also the content of the peptide segment AP in the corn peptide can be ≥8.3 mg / 100 g, the content of the peptide segment IP can be ≥33.95 mg / 100 g, and the content of the peptide segment VAYPE can be ≥4.26 mg / 100 g. The preparation method of the present application is simple and fast in operation and is convenient for large-scale production.

[0058] In a specific embodiment, the enzyme activity of the alkaline protease is 2.4 AU and the enzyme activity of the bromelain is 50,000-100,000 U per gram of corn protein.

[0059] The applicant found in the research of the enzymatic treatment that when the enzyme activity of the alkaline protease and the bromelain meets the above range, it is beneficial to further exert the effect of the enzyme preparation and improve the mass content of the functional peptide segments in the corn peptide. Specifically, the alkaline protease can be the Alcalase 2.4L type alkaline protease of Novozymes.

[0060] Further, the applicant also found that the temperature of the first enzymolysis is 50-65℃, the time is 20-45 min, and the pH value is 8.5, which is helpful to better exert the enzymolysis effect of the alkaline protease; the temperature of the second enzymolysis is 40-60℃, the time is 110-130 min, and the pH value is 7, which is helpful to better exert the enzymolysis effect of the bromelain.

[0061] Specifically, the corn protein solution can be first adjusted to the optimal enzymolysis pH environment of the alkaline protease, i.e. the pH value is 8.5, to obtain the enzymolysis stock solution which is beneficial to the first enzymolysis of the alkaline protease. Specifically, sodium hydroxide NaOH or hydrochloric acid HCl can be used for the adjustment. Subsequently, under the suitable enzymolysis temperature of the alkaline protease, i.e. 50-65℃, the alkaline protease is added to the enzymolysis stock solution, and then the enzymolysis is performed for 20-45 min, so that the alkaline protease acts on the protein in the enzymolysis stock solution, fully opens the high-level structure of the protein to expose more enzyme cutting sites, thereby obtaining the first enzymolysis solution. To maximize the enzymolysis efficiency of the bromelain, the pH value of the first enzymolysis solution can be adjusted to the optimal enzymolysis pH environment of the bromelain, i.e. the pH value is 7, before the bromelain is added for the second enzymolysis. Specifically, sodium hydroxide NaOH or hydrochloric acid HCl can be used for the adjustment. Under the suitable enzymolysis temperature of the bromelain, i.e. 40-60℃, the bromelain is added to the first enzymolysis solution with the pH value of 7, and then the enzymolysis is performed for 110-130 min, so that the bromelain fully acts on the protein in the first enzymolysis solution, thereby obtaining the second enzymolysis solution.

[0062] In addition, after the enzymolysis treatment is completed, the enzyme preparation can be subjected to enzyme inactivation treatment to lose the catalytic activity. The present application does not limit the enzyme inactivation method, and the enzyme inactivation can be performed by using the conventional enzyme inactivation means in the art, such as heating to 95℃ for 30 min.

[0063] The second enzymatic hydrolysate obtained after enzyme inactivation can be subjected to centrifugation to collect the supernatant for subsequent separation and purification. Specifically, the centrifugation speed can be 2500-3500 rmp / min, and the centrifugation time can be 5-10 min.

[0064] In a specific embodiment, the separation and purification process comprises sequentially filtering, activated carbon adsorption, cation exchange resin treatment, and anion exchange resin treatment of the second enzymatic hydrolysate.

[0065] The filtering process can further improve the mass content of the functional peptide segment, and retain components with a larger molecular weight to clarify the enzymatic hydrolysate. The activated carbon adsorption process can remove impurities and odors from the product. The cation exchange resin treatment can cause adsorption and exchange reactions between the enzymatic hydrolysate and cations in the system to separate and purify the enzymatic hydrolysate. The anion exchange resin treatment can cause adsorption and exchange reactions between the enzymatic hydrolysate and anions in the system to further separate and purify the enzymatic hydrolysate.

[0066] Specifically, the filtering process comprises filtering with a filter membrane having a pore size of 200 nm.

[0067] It can be understood that after the filtering process, substances with a particle size of ≥200 nm will be removed, and this step can clarify the enzymatic hydrolysate and further enrich the functional peptide segment.

[0068] The activated carbon adsorption process comprises using activated carbon with a particle size of ≤0.075 mm for activated carbon adsorption treatment at a temperature of 40-60°C for 30-40 min.

[0069] Using activated carbon with a particle size of ≤0.075 mm can better remove odors. To further improve the activated carbon adsorption capacity, the temperature for activated carbon adsorption treatment is limited to 40-60°C, and the time is limited to 30-40 min. It can be understood that after the activated carbon adsorption treatment is completed, the activated carbon needs to be removed.

[0070] In addition, the cation exchange resin treatment specifically comprises passing the activated carbon-adsorbed enzymatic hydrolysate through a cation exchange resin column at a linear flow rate of 1 mL / min, collecting the effluent when the ultraviolet detection value reaches 100 mAu, and stopping collecting the effluent when the ultraviolet detection value is less than 200 mAu.

[0071] The anion exchange resin treatment specifically includes: passing the effluent collected in the cation exchange resin purification through an anion exchange resin column at a linear flow rate of 1 mL / min, starting to collect the effluent when the ultraviolet detection value reaches 100 mAu, and stopping collecting the effluent when the ultraviolet detection value is lower than 200 mAu, and the effluent collected after the anion exchange resin purification is the purified product after separation and purification treatment.

[0072] Since the effluent flow rate is relatively stable when the ultraviolet detection value reaches 100 mAu at the beginning of the treatment, the present application limits the collection of the effluent when the ultraviolet detection value reaches 100 mAu; since the content of the target corn peptide in the effluent is very low when the ultraviolet detection value is lower than 200 mAu, the collection of the effluent is stopped, which can further enrich the functional peptide segment.

[0073] The third aspect of the present application provides a use of the above corn peptide in the preparation of an anti-tumor related product. It can be understood that the product includes but is not limited to food, health care products and drugs. A large amount of research data proves that the corn peptide with the content of peptide segment AP≥8.3 mg / 100 g, the content of peptide segment IP≥33.95 mg / 100 g, and the content of peptide segment VAYPE≥4.26 mg / 100 g has a significant anti-tumor ability, which can not only be applied to conventional food, health care products, such as liver-protecting food or liver-protecting products, but also can be used for preparing anti-tumor drugs, thereby widening the application range of corn peptides and providing new raw materials for anti-tumor drugs.

[0074] Hereinafter, the technical solutions of the present application will be further explained and described in combination with specific examples. The experimental methods in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturers, unless otherwise specified. The reagents used, if not specifically stated, are commercially available or can be obtained from public channels.

[0075] Example 1: Preparation of corn peptide

[0076] (1) Enzymatic treatment

[0077] 300 g of corn meal was mixed with 3 L of deionized water and stirred at a stirring speed of 300 rpm / min for 40 min. Then, 2.7 g of Alcalase 2.4L alkaline protease of Novozymes Company was added to the mixture for enzymatic hydrolysis at a pH of 8.5 and a temperature of 55℃ for 40 min. Then, 1.5 g of bromelain was added to the mixture for enzymatic hydrolysis at a pH of 7 and a temperature of 45℃ for 120 min. After the two-step enzymatic hydrolysis, the mixture was subjected to enzyme inactivation at 95℃ for 30 min to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged at a centrifugal speed of 3000 rpm / min for 10 min, and the supernatant was collected for subsequent processes.

[0078] (2) Separation and purification treatment

[0079] The separation and purification treatment included filtration, activated carbon adsorption, cation exchange resin purification, and anion exchange resin purification. First, the enzymatic hydrolysate was filtered by using a filter membrane with a pore size of 200 nm. Then, the filtered enzymatic hydrolysate was subjected to activated carbon adsorption by using food-grade activated carbon with a particle size of less than 0.0750 mm at a temperature of 45℃ for 35 min, and then the activated carbon was removed. The enzymatic hydrolysate after the activated carbon adsorption was passed through a cation exchange resin column (LX-010, Shanghai Suner Chemical Technology Co., Ltd.) at a linear flow rate of 1 mL / min. When the ultraviolet detection value reached 100 mAu, the effluent was collected. When the ultraviolet detection value was lower than 200 mAu, the collection of the effluent was stopped. The effluent collected in the cation exchange resin purification was passed through an anion exchange resin column (D001H, Jiangsu Suqing Water Treatment Engineering Group Co., Ltd.) at a linear flow rate of 1 mL / min. When the ultraviolet detection value reached 100 mAu, the effluent was collected. When the ultraviolet detection value was lower than 200 mAu, the collection of the effluent was stopped. The effluent collected after the anion exchange resin purification was the purified product after the separation and purification treatment.

[0080] (3) Spray drying treatment

[0081] The purified product was subjected to spray drying treatment at an inlet temperature of 160℃ and an outlet temperature of 65℃. After the spray drying treatment, a powdery corn peptide product was obtained.

[0082] Example 2: Quantification of corn peptides

[0083] (1) Identification of corn peptides

[0084] A corn peptide solution of 20 pg / mL was prepared by adding purified water to the corn peptide product obtained in Example 1, which was the sample to be detected. An Inertsil ODS-3 column was connected to a liquid chromatograph, and the injection volume was set to 5 pL. In the positive ion mode, the collision energy was set to -25 V and -35 V, and product ion scanning (PIS) was performed on the sample to be detected to obtain product ion mass spectra, which can be seen in FIGS. 1-3. FIG. 1 is the product ion mass spectrum of the peptide segment AP, FIG. 2 is the product ion mass spectrum of the peptide segment IP, and FIG. 3 is the product ion mass spectrum of the peptide segment VAYPE. In FIGS. 1-3, panel A is the product ion mass spectrum obtained by setting the collision energy to -25 V, and panel B is the product ion mass spectrum obtained by setting the collision energy to -35 V.

[0085] The liquid chromatography conditions were as follows: mobile phase A was purified water (containing 0.1% (v / v) formic acid), mobile phase B was 100% acetonitrile (containing 0.1% (v / v) formic acid), and the flow rate was 0.2 mL / min. The gradient elution program specifically included: the first gradient elution time was 0 min-15 min, and the proportion of mobile phase B was 0%-40%; the second gradient elution time was 15 min-20 min, and the proportion of mobile phase B was 40%-80%; the third gradient elution time was 20 min-25 min, and the proportion of mobile phase B was 80%; the fourth gradient elution time was 25.1 min-35 min, and the proportion of mobile phase B was 0%. The column oven was 40°C.

[0086] The mass spectrometry conditions were as follows: the ionization mode was electrospray ionization (ESI), the ion spray voltage was 4.5 kV, the flow rate of the atomization gas (nitrogen) was 3.0 L / min, the flow rate of the heating gas (air) was 10 L / min, the flow rate of the drying gas (nitrogen) was 10 L / min, the desolvation tube temperature was 250°C, the heating module temperature was 400°C, and the ion source temperature was 300°C.

[0087] Based on the above product ion mass spectra, the sequence structure of the peptide segment was determined in combination with the NCBI database and the UniProt database, and three target peptide segments were identified from the corn peptide, namely Ala-Pro (AP), Ile-Pro (IP), and Val-Ala-Tyr-Pro-Glu (VAYPE), as shown in Table 1. Based on the precursor ion information, PIS scanning was performed on the corn peptide. If there is product ion information corresponding to a peptide segment with certain amino acid composition after analysis of the product ion mass spectrum, it indicates that the corn peptide contains the corresponding peptide segment structure.

[0088] Table 1

[0089] (2) Content determination of target peptide segments in corn peptides

[0090] The multiple reaction monitoring (MRM) technology in high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to optimize the quantitative ion pair and voltage of the standard peptide segments, and the MRM optimization conditions were obtained, as shown in Table 2.

[0091] The retention time of the three standard peptide segments AP, IP and VAYPE was determined by using the MRM optimization conditions, and the three standard peptide segments were mixed and diluted with water to obtain a series of standard working solutions with concentrations of 1.95 μg / L, 3.91 μg / L, 7.81 μg / L, 15.63 μg / L, 31.25 μg / L, 62.5 μg / L, 125 μg / L, 250 μg / L, 500 μg / L and 1000 μg / L, respectively. The injection volume was set to 10 μL, and the standard curve was drawn under the optimized conditions, with the mass concentration (X, μg / L) as the abscissa and the peak area (Y) as the ordinate. The standard curve of the peptide segments AP, IP and VAYPE is shown in FIGS. 4-6, and the corresponding standard curve equations are shown in Table 3.

[0092] The corn peptide solution with a concentration of 20 μg / mL obtained in Example 2 was diluted 100 times with pure water, centrifuged at 10000 rpm for 10 min, and the supernatant was filtered with a nylon filter membrane with a pore size of 0.22 μm to obtain the sample to be detected. The peak area external standard method was used to determine the content of the target peptide segments in the corn peptide according to the standard curve, as shown in Table 4. The standard peptide segments corresponding to the target peptide segments and the quantitative chromatograms of the target peptide segments are shown in FIGS. 7-9. FIG. 7 is the quantitative chromatogram of the target peptide segment of the peptide segment AP, FIG. 8 is the quantitative chromatogram of the target peptide segment of the peptide segment IP, and FIG. 9 is the quantitative chromatogram of the target peptide segment of the peptide segment VAYPE. In FIGS. 7-9, A is the chromatogram of the standard peptide segment, and B is the quantitative chromatogram of the corresponding target peptide segment of the corn peptide.

[0093] The liquid chromatography conditions are as follows: the chromatographic column is Inertsil ODS-3 (particle size is 5 μm, inner diameter is 2.1 mm, column length is 250 mm); the injection volume is 10 μL; the mobile phase A is pure water (containing 0.1% (v / v) formic acid), and the mobile phase B is 100% acetonitrile (containing 0.1% (v / v) formic acid), and the flow rate is 0.2 mL / min. The gradient elution program specifically includes: the first gradient elution time is 0 min-15 min, the mobile phase B accounts for 0%-50%; the second gradient elution time is 15 min-20 min, the mobile phase B accounts for 50%-100%; the third gradient elution time is 20 min-25 min, the mobile phase B accounts for 100%; the fourth gradient elution time is 25.1 min-35 min, and the mobile phase B accounts for 0%. The column oven is 40℃.

[0094] The mass spectrometry conditions are as follows: the ionization mode is ESI, and the positive ion mode; the ion spray voltage is +4.5 kV, the flow rate of atomization gas (nitrogen) is 3.0 L / min, the flow rate of heating gas (nitrogen) is 10 L / min, the flow rate of dry gas (nitrogen) is 10 L / min, the desolvation tube temperature is 250℃, the heating module temperature is 400℃, and the ion source temperature is 300℃; the scanning mode is MRM, the residence time is 100 ms, and the delay time is 3 ms.

[0095] The results show that the contents of the target peptide segments of the corn peptide are IP>AP>VAYPE from high to low.

[0096] Table 2

[0097] Table 3

[0098] Table 4

[0099] (3) Corn peptide product determination

[0100] The components content and molecular weight distribution of the corn peptide product in Example 1 are detected by the national standard method, wherein the detection method of protein content is GB 5009.5, the detection method of total amino acid content and free amino acid content is GB 5009.124, the detection method of oligopeptide content is GB 5009.5 and GB 5009.124, and the detection method of molecular weight is GB / T 22729.

[0101] The detected protein content in the corn peptide (dry basis) was 84.47 g / 100 g, the total amino acid content was 80.56 g / 100 g, and the free amino acid content was 3.74 g / 100 g. The gel chromatogram of the molecular weight distribution is shown in Figure 10, where λ = 220 nm. According to the peak area percentage of Figure 10, the content of peptide segments above 10,000 u was 0%, the content of peptide segments between 5,000-1,000 u was 0%, the content of peptide segments between 2,000-5,000 u was about 0.209%, the content of peptide segments between 1,000-2,000 u was about 1.5722%, the content of peptide segments between 189-1,000 u was about 49.61%, the content of peptide segments between 132-189 u was about 15.8606%, and the content of peptide segments between 1-132 u was about 32.4924%. Therefore, the proportion of hydrolysate with a relative molecular mass less than 1,000 u was about 97.96%.

[0102] Example 3: Functional experiment of corn peptide

[0103] (1) Cell toxicity of corn peptide

[0104] The corn peptide product obtained in Example 1 was added to a serum-free medium to prepare a serum-free medium containing corn peptide with a final concentration of 0 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, and 10 mg / mL. Human tumor cells JHH7 cells were cultured in the medium for 24 hours to obtain corn peptide pretreated JHH7 cells. The cell viability of the above-mentioned JHH7 cells was detected by MTT method to evaluate the effect of corn peptide with different final concentrations on the cell viability of JHH7 cells. The group with a final concentration of 0 mg / mL of corn peptide was set as the Blank group.

[0105] The MTT detection results are shown in Figure 11. At a final concentration of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL, the cell viability of corn peptide pretreated JHH7 cells after 24 hours had no significant difference compared with the Blank group. The cell viability of JHH7 cells pretreated with corn peptide at a concentration of 1 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, and 10 mg / mL had statistical significance compared with the Blank group, which could cause a slight increase in cell viability. To ensure the best growth conditions for subsequent experiments, the corn peptide concentrations selected for subsequent experiments were determined to be 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL.

[0106] (2) Ability of corn peptide to scavenge intracellular reactive oxygen species (ROS)

[0107] JHH7 cells were treated with ethanol (EtOH) at a final concentration of 500 μM for 6 h. Subsequently, the corn peptide product obtained in Example 1 was added to the serum-free medium to prepare serum-free medium containing corn peptides at final concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL and 0.8 mg / mL, respectively. The JHH7 cells treated with ethanol were cultured in the serum-free medium for 24 h to obtain corn peptide-treated JHH7 cells. The above cells were treated with fluorescent dye DCFH-DA at a final concentration of 10 μM and incubated at 37 °C for 30 min to promote the uptake of the probe by the cells. After incubation, the cells were washed twice with phosphate buffered saline (PBS) to eliminate any remaining extracellular DCFH-DA probe. The cells were trypsinized, suspended in PBS and analyzed for the mean fluorescence intensity of DCFH-DA, which reflects the level of intracellular ROS, using a flow cytometer. The excitation / emission wavelength of DCFH-DA was 485 / 530 nm.

[0108] wherein the JHH7 cells without any treatment were set as the Blank group; the JHH7 cells treated with EtOH at a final concentration of 500 μM for 6 h were set as the Model group; and the JHH7 cells treated with EtOH at a final concentration of 500 μM for 6 h and then treated with corn peptides at different final concentrations were set as the Cp groups.

[0109] As shown in Figure 12, it was found that the ROS level of the Model group was significantly increased by about 140.27% (p < 0.05) compared with the Blank group. The treatment of JHH7 cells with corn peptides at different final concentrations resulted in a dose-dependent decrease in the ROS level, wherein the Cp group with a final concentration of 0.8 mg / mL could effectively reduce the ROS level below the normal level, indicating that corn peptides had a significant ROS scavenging capacity and further supporting the potential antioxidant properties and the ability to alleviate oxidative stress of corn peptides. Since there is a complete oxidation-antioxidation system in the body, the ROS can be maintained within a stable range under normal circumstances, but when the balance is broken, the ROS will continue to rise, which will promote cell transformation and lead to the occurrence of malignant tumors. Therefore, the ROS scavenging capacity of corn peptides indicates that corn peptides also have the potential to resist tumors.

[0110] (3) Effect of corn peptides on the intracellular calcium ion (Ca2+) concentration and mitochondrial membrane potential (ΔΨm) 2+

[0111] ​After the JHH7 cells were incubated at 37°C for 24 hours, the culture medium was aspirated, and the JHH7 cells were washed with PBS buffer to remove the residual culture medium and debris. Subsequently, the corn peptide product obtained in Example 1 was added to the 2% serum supplemented medium to prepare 2% serum supplemented medium containing corn peptides at final concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL and 0.8 mg / mL, respectively, and the above JHH7 cells were cultured with the medium for 24 hours. The intracellular Ca 2+ concentration and mitochondrial transmembrane potential were detected using the calcium ion activity kit and mitochondrial transmembrane potential assay kit produced by Biyun Tian Biotechnology Research Institute, respectively. Among them, the JHH7 cells without corn peptide treatment were set as the Blank group; and the JHH7 cells treated with different final concentrations of corn peptides were set as the Cp groups.

[0112] As shown in FIG. 13, for the intracellular Ca 2+ concentration, it can be found that the fluorescence intensity of all Cp groups pretreated with corn peptides (0.1-0.8 mg / mL) increased significantly and in a dose-dependent manner (p < 0.05). This indicates that corn peptides have a significant effect on the intracellular Ca 2+ concentration. The increase in intracellular Ca 2+ concentration leads to the redistribution of Ca 2+ in the cytoplasm, indicating that corn peptides can regulate the intracellular Ca 2+ concentration, thereby affecting cell function and signaling pathways. The endoplasmic reticulum and mitochondria are important organelles that store Ca 2+ and regulate calcium homeostasis in cells, and can transport a large amount of Ca 2+ into the cytoplasm. Among them, mitochondria not only serve as the energy supply center of cells, but also maintain intracellular calcium homeostasis and are very important in intracellular Ca 2+ metabolism. At the same time, Ca 2+ plays a crucial role in various physiological and biochemical reactions of life activities and the occurrence and development of diseases. Ca 2+ has the ability to promote mitosis and stimulate cell proliferation, and is closely related to the growth and proliferation of tumor cells. Corn peptides can regulate the intracellular calcium level, indicating that they may further affect the growth and proliferation of tumor cells.

[0113] Mitochondrial membrane potential is crucial for ATP synthesis and cell survival. For mitochondrial membrane potential, it was found that the fluorescence intensity of all Cp groups was significantly lower than that of the Blank group (p<0.05) and had a dose-dependent effect, which indicated that corn peptides could significantly reduce the mitochondrial transmembrane potential of tumor cells. Since the transmembrane potential of mitochondria would decrease due to mitochondrial damage and lack of energy, it could further prove that corn peptides could affect the function of tumor cell mitochondria and had a positive anti-tumor effect. Mitochondria absorb Ca 2+ The energy source for mitochondria is the △Ψm formed by mitochondrial respiratory metabolism, so the decrease in mitochondrial membrane potential will affect the influx of Ca 2+ into mitochondria; at the same time, the decrease in membrane potential will also lead to changes in mitochondrial membrane permeability, causing the opening of mitochondrial membrane permeability transition pores, which in turn promotes the leakage of Ca 2+ in mitochondria into the cytoplasm, leading to an increase in the intracellular (i.e., cytoplasmic) Ca 2+ concentration.

[0114] (4) Corn peptides affect the expression of key protein-related mRNA and key proteins in cells

[0115] JHH7 cells were pretreated with corn peptides at final concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, and 0.8 mg / mL, and the RNA of the above JHH7 cells was extracted using a total RNA extraction kit and reverse transcribed into cDNA. Subsequently, the expression levels of key protein-related mRNA in corn peptide-pretreated JHH7 cells were evaluated using reverse transcription polymerase chain reaction (RT-PCR); and the expression levels of key proteins involved in cell processes in the above corn peptide-pretreated JHH7 cells were evaluated using Western blotting, and the key proteins specifically included inositol 1,4,5-trisphosphate receptor (IP3R), calreticulin, human chromosome 10-deleted phosphatase (PTEN), cyclin-dependent kinase inhibitor 1 (P21), cyclin-dependent kinase inhibitor (P27), proliferating cell nuclear antigen (PCNA), AKT serine / threonine kinase 1 (Akt1), and AKT serine / threonine kinase 2 (Akt2). Among them, the above corn peptide-pretreated JHH7 cells were set as the Cp group, and the JHH7 cells without corn peptide pretreatment were set as the Blank group.

[0116] For mRNA expression levels, as shown in Table 5, the mRNA expression levels of IP3R, Calreticulin, PTEN, P21 and P27 in the Cp group were significantly increased (p<0.05); the mRNA expression levels of PCNA, Akt1 and Akt2 in the 0.2-0.8 mg / mL Cp group were significantly decreased (p<0.05). Among the different concentrations tested, the 0.4 mg / mL concentration showed the most obvious intervention effect, showing a significant difference compared with other concentrations (p<0.05). The results showed that when corn peptides were administered at a concentration of 0.4 mg / mL, they could significantly inhibit the mRNA expression levels of PCNA, Akt1 and Akt2 in JHH7 cells, showing a strong inhibitory effect.

[0117] Table 5

[0118] For protein expression levels, as shown in Figures 14-16, Figure 14 is a columnar result graph of the detection of the protein expression levels of IP3R, Calreticulin, PTEN, P21, P27 in cells, Figure 15 is a columnar result graph of the detection of the protein expression levels of PCNA, Akt1 and Akt2 in cells, and Figure 16 is a protein immunoblotting graph of IP3R, Calreticulin, PTEN, P21, P27, Akt1 and Akt2 in cells. Corn peptide pretreatment can cause the protein expression levels of IP3R, Calreticulin, PTEN, P21 and P27 to increase and the protein expression levels of PCNA, Akt1 and Akt2 to decrease. Among them, corn peptides showed the most effective effect in enhancing PTEN protein expression, followed by P27. The results showed that corn peptides have the ability to regulate the protein expression of key regulatory molecules involved in cell proliferation and signal pathways. In addition, corn peptides showed the most obvious effect in reducing PCNA expression, followed by Akt1 and Akt2.

[0119] IP3R is a transmembrane protein of the endoplasmic reticulum. When IP3 in the cytoplasm binds to IP3R on the endoplasmic reticulum membrane, it can release Ca 2+ in the endoplasmic reticulum Ca 2+Concentration; Calreticulin is an important calcium-binding protein located in the endoplasmic reticulum, which plays a crucial role in maintaining intracellular calcium homeostasis and promoting intracellular calcium signal transmission. Overexpression of Calreticulin can lead to increased uptake of calcium into the endoplasmic reticulum, thereby affecting the potential energy of calcium and the integrity of the mitochondrial membrane, which indicates that Calreticulin may affect cell processes regulated by calcium signaling and mitochondrial function; PCNA is a nuclear protein that is a key marker for assessing the level of cell proliferation. The expression rate of PCNA decreases, and the number of proliferative cells decreases. 2+ Calcium is an important second messenger in cells, and changes in cytoplasmic calcium concentration will affect cell gene transcription through signal transduction systems.

[0120] The PTEN / Akt pathway is closely related to intracellular calcium signaling. PTEN is located on human chromosome 10q23 and is a tumor suppressor gene that plays a crucial role in regulating cell processes, including cell growth, proliferation, and survival. PTEN controls cell survival and proliferation by inhibiting Akt activation. Dysregulation of the PTEN / Akt pathway is common in various cancers and is associated with tumor development and progression. After dephosphorylation and activation of PTEN, it has a dual role as a protein and lipid phosphatase. The protein phosphatase action of PTEN can remove the phosphate on tyrosine, which may act on the same substrate as many cancer proteins to inhibit tumors. Enhancement of PTEN function inhibits the transmission of cell proliferation signals, which will inactivate downstream Akt. Akt is a serine-threonine kinase that is a downstream effector molecule in the PI3K signaling pathway; the PI3K pathway is a key signaling pathway involved in various cellular processes, including apoptosis, senescence, and proliferation. PTEN and Akt are two balancing factors that act in opposite directions. When PTEN is enhanced and Akt is not activated, P21 and P27 downstream of the pathway will be activated to inhibit the cell cycle and resist cell proliferation. Reduced or absent P21 expression reduces the inhibitory effect on cell proliferation, which can lead to abnormal cell proliferation and differentiation and increase the risk of tumor occurrence. P27 is an important member of the KIP family of two cyclin-dependent kinase inhibitors (CKI) families (KIP family and INK4 family), which can inhibit the activity of various cyclin-CDK complexes, have a negative effect on the cell cycle, and can regulate, inhibit cell division and proliferation, and promote cell differentiation and apoptosis. In summary, corn peptides exhibit strong ability to regulate the PTEN / Akt signaling pathway in JHH7 cells. Therefore, the increase in expression of tumor suppressor genes and the decrease in expression of oncogenes contribute to the anti-tumor effect, and by regulating this important signaling pathway, corn peptides have the potential to disrupt tumor cell growth and promote favorable cell outcomes in the context of cancer treatment.

[0121] In summary, the anti-tumor effect of corn peptides was evaluated using JHH7 cell model. Corn peptides have the ability to eliminate reactive oxygen species in JHH7 cells, reduce mitochondrial membrane potential, increase free calcium, and enhance the expression of calnexin mRNA and protein, which in turn can affect the calcium homeostasis of tumor cells. In addition, corn peptides can up-regulate the mRNA and protein expression of tumor suppressor genes PTEN, P21 and P27, and down-regulate the expression of oncogene Akt. Therefore, the anti-tumor effect of corn peptides may be through affecting the mitochondrial membrane permeability of tumor cells, releasing calcium ions stored in mitochondria into the cytoplasm. The ability of tumor cells to regulate calcium homeostasis in the cytoplasm gradually decreases with the damage of mitochondria, further exacerbating the accumulation of calcium in the cytoplasm. The concentration change of Ca 2+ as a second messenger will affect the signal transduction system of tumor cells, and through multiple intracellular transduction processes, the expression of tumor suppressor genes in tumor cells will increase, and the expression of oncogenes will decrease, ultimately achieving an anti-tumor effect.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A corn peptide having an antitumor effect, wherein, The corn peptide is composed of at least peptide segments AP, IP and VAYPE. Based on the mass of the corn peptide, the content of the peptide segment AP is ≥8.3 mg / 100 g, the content of the peptide segment IP is ≥33.95 mg / 100 g, and the content of the peptide segment VAYPE is ≥4.26 mg / 100 g.

2. The corn peptide of claim 1, wherein, The content of protein in the corn peptide is ≥84.47 g / 100 g, the content of amino acid is ≥80.56 g / 100 g, and the mass content of peptide with a molecular weight less than 1000 u is ≥97%.

3. The corn peptide according to claim 1 or 2, wherein, The corn peptide is obtained by sequentially subjecting corn protein to enzymatic treatment and separation and purification treatment. The enzymatic treatment comprises using alkaline protease and bromelain for enzymatic hydrolysis.

4. A method of preparing the corn peptides of any one of claims 1-3, wherein, The method comprises the following steps: 1) subjecting corn protein to first enzymatic hydrolysis using alkaline protease to obtain a first enzymatic hydrolysate, and subjecting the first enzymatic hydrolysate to second enzymatic hydrolysis using bromelain to obtain a second enzymatic hydrolysate; 2) subjecting the second enzymatic hydrolysate to separation and purification treatment to obtain the corn peptide.

5. The production method according to claim 4, wherein, Based on 1 g of the corn protein, the enzyme activity of the alkaline protease is 2.4 AU, and the enzyme activity of the bromelain is 50000-100000 U.

6. The production method according to claim 4 or 5, wherein, The first enzymatic hydrolysis is performed at a temperature of 50-65 °C for 20-45 min at a pH of 8.5; and / or The second enzymatic hydrolysis is performed at a temperature of 40-60 °C for 110-130 min at a pH of 7.

7. The method of making according to any one of claims 4-6, wherein, The separation and purification treatment comprises sequentially subjecting the second enzymatic hydrolysate to filtration treatment, activated carbon adsorption treatment, cation exchange resin treatment and anion exchange resin treatment.

8. The production method according to claim 7, wherein The filtration treatment comprises using a filter membrane with a pore size of 200 nm for filtration treatment.

9. The production method according to claim 7 or 8, wherein The activated carbon adsorption treatment comprises using activated carbon with a particle size of ≤0.075 mm for activated carbon adsorption treatment at a temperature of 40-60 °C for 30-40 min.

10. Use of the corn peptide of any one of claims 1-3 in the preparation of an anti-tumor related product.

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