Application of corn germ concentrated powder in improvement of osteoarthritis

Abstract

The invention belongs to the technical field of food and medicine, and relates to application of corn germ concentrated powder in improvement of osteoarthritis. The invention particularly relates to application of corn germ concentrated powder, namely corn antioxidant multienzyme, in preparation of medicines or health-care foods for treating rheumatoid arthritis and osteoporosis. In the corn germ concentrated powder, superoxide dismutase / (U / g) is greater than or equal to 8500, glutathione peroxidase / (U / g) is greater than or equal to 5000, and catalase / (U / g) is greater than or equal to 750. The corn germ concentrated powder can also be prepared by the following method: cleaning corn, germinating, adding water into the germinated corn, grinding and pulping, adding a settling agent into the corn steep liquor, standing, taking supernate, adding part of sodium ascorbate, performing temperature-controlled concentration, adding the rest sodium ascorbate and a stabilizer into the concentrated solution, and uniformly mixing to obtain the corn germ concentrated powder. And drying at controlled temperature, crushing, and mixing with a proper amount of silicon dioxide. The prepared corn germ concentrated powder has the effect of resisting rheumatoid arthritis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food and medicine, and relates to application of corn germ concentrate powder in improvement of osteoarthritis.

[0002] In particular, the application relates to application of corn germ concentrate powder in preparation of medicines for treating rheumatoid arthritis and osteoporosis and foods for improving bone density. BACKGROUND

[0003] Rheumatoid arthritis is a kind of autoimmune disease with refractory, progressive and systemic characteristics, which is mainly characterized by invasion of synovial chronic inflammation and joints, and seriously endangers human health. Meanwhile, there is a close correlation between osteoporosis and rheumatoid arthritis. Rheumatoid arthritis patients usually face a higher risk of osteoporosis, which is mainly caused by bone loss due to inflammation, use of glucocorticoids and limited activity. The systemic inflammatory process of rheumatoid arthritis can cause increased bone resorption and decreased bone formation, thereby accelerating bone loss. The use of glucocorticoids is an important risk factor for osteoporosis in patients with rheumatoid arthritis, and long-term use can exacerbate bone loss. Due to joint pain and limited function, the activity of patients with rheumatoid arthritis is reduced, which also leads to reduced bone density. Therefore, the treatment plan for rheumatoid arthritis should consider the related factors of rheumatoid arthritis and osteoporosis, and optimize the treatment of rheumatoid arthritis. Existing rheumatoid arthritis drugs, including traditional synthetic drugs (such as methotrexate, hydroxychloroquine, sulfadiazine), targeted synthetic drugs (such as JAK inhibitors) and biological agents (such as TNF-α inhibitors), can effectively improve symptoms and prevent disease progression, but are accompanied by significant side effects and high costs. Methotrexate, for example, shows efficacy in short-term and long-term studies, but it can cause severe liver and lung toxicity, and it is difficult to achieve the best therapeutic dose in some patients. In addition, many patients have poor tolerance to high doses of methotrexate, and even the use of folic acid supplements is difficult to improve. Therefore, it is urgent to develop new safe and effective drugs with the effect of preventing or treating osteoporosis.

[0004] The corn germ concentrate powder is prepared by germination, beating, and sedimentation of corn as raw material, and then adding auxiliary materials.

[0005] The corn germ concentrate powder has the functions of reducing blood pressure, immune regulation, antioxidant and liver protection. It has the effects of clearing the intestinal tract, clearing toxins, clearing free radicals, resisting sugar, resisting cell aging, nourishing the heart and calming the nerves, and improving sleep quality. Currently, there is no related report on its application in improving osteoarthritis, treating rheumatoid arthritis and the like. SUMMARY

[0006] The technical problem solved by the present application is to provide a corn germ concentrate and a preparation method thereof.

[0007] The corn germ concentrate is obtained by germination, pulping, impurity removal, concentration and drying of corn.

[0008] The corn germ concentrate can be prepared by the following method: The corn is washed and germinated, water is added to the germinated corn for grinding and pulping, a settling agent is added to the corn slurry for standing, the supernatant is taken, part of the sodium ascorbate is added for temperature-controlled concentration, the remaining sodium ascorbate and stabilizer are added to the concentrated solution for temperature-controlled drying, crushing, and mixing with the appropriate amount of silicon dioxide.

[0009] The germination length is 4-12mm, preferably 4-8mm; The grinding time is 1-3 hours, preferably 3 hours; The sodium ascorbate accounts for 15-25% of the corn germ concentrate; The mass ratio of the first added sodium ascorbate to the remaining sodium ascorbate is 1:1-1.5, preferably 1:1; The mass ratio of the corn to the settling agent is 10:2.5-3.5; The settling agent is a mixture of calcium chloride and kaolin, and the mass ratio of calcium chloride to kaolin is 1:0.5-1.5, preferably 1:1; The stabilizer is a mixture of malt dextrin and beta-cyclodextrin, and the mass ratio of malt dextrin to beta-cyclodextrin is 2-3:1-2; The stabilizer accounts for 8-12% of the mass percentage of the corn germ concentrate; The mass-volume ratio of the corn to water is 1:3-5.

[0010] The temperature for temperature-controlled concentration and temperature-controlled drying is 60-70℃. The temperature-controlled concentration is 25-35min.

[0011] Preferably, the corn germ concentrate can be prepared by the following method: The corn is washed and germinated, 3-5 times water is added to the germinated 4-8mm corn for grinding and pulping, a settling agent is added to the corn slurry for standing, the supernatant is taken, part of the sodium ascorbate is added for temperature-controlled concentration, the remaining sodium ascorbate and stabilizer are added to the concentrated solution for temperature-controlled drying, crushing, and mixing with the appropriate amount of silicon dioxide. The settling agent is a mixture of calcium chloride and kaolin, and the mass ratio of calcium chloride to kaolin is 1:1; The mass ratio of the corn to the settling agent is 10:3; The stabilizer is a mixture of malt dextrin and beta-cyclodextrin, wherein the mass ratio of malt dextrin to beta-cyclodextrin is 2:1. The sodium ascorbate accounts for 20% of the corn germ concentrate powder. The mass ratio of the first added sodium ascorbate to the remaining sodium ascorbate is 1:1. The stabilizer accounts for 10% of the mass percentage of the corn germ concentrate powder.

[0012] In the corn germ concentrate powder prepared by the method, the superoxide dismutase is (U / g) ≥8500, the glutathione peroxidase is (U / g) ≥5000, and the catalase is (U / g) ≥750. Further, the superoxide dismutase is (U / g) ≥9036, the glutathione peroxidase is (U / g) ≥6524, and the catalase is (U / g) ≥835.

[0013] Further, the superoxide dismutase is (U / g) ≥10000, the glutathione peroxidase is (U / g) ≥7200, and the catalase is (U / g) ≥1200.

[0014] Further, the superoxide dismutase is (U / g) ≥11964, the glutathione peroxidase is (U / g) ≥8202, and the catalase is (U / g) ≥1366.

[0015] It can be seen that the preparation method can significantly improve the enzyme activity of antioxidant enzymes in the corn germ concentrate powder, and can significantly improve the activity of superoxide dismutase, glutathione peroxidase and / or catalase.

[0016] The application provides application of the corn germ concentrate powder in preparation of a medicine for improving osteoarthritis.

[0017] The application provides application of the corn germ concentrate powder in preparation of a medicine for treating rheumatoid arthritis.

[0018] The rheumatoid arthritis is rheumatoid arthritis stimulated by tumor necrosis factor alpha.

[0019] The corn germ concentrate powder plays a role in treating rheumatoid arthritis by inhibiting tumor necrosis factor alpha.

[0020] The application provides application of the corn germ concentrate powder in preparation of a food for helping to improve bone density.

[0021] In the corn germ concentrate powder, the superoxide dismutase is (U / g) ≥8500, the glutathione peroxidase is (U / g) ≥5000, and the catalase is (U / g) ≥750.

[0022] Further, the corn germ concentrate powder has superoxide dismutase (U / g) ≥9036, glutathione peroxidase (U / g) ≥6524, and catalase (U / g) ≥835.

[0023] Further, the corn germ concentrate powder has superoxide dismutase (U / g) ≥10000, glutathione peroxidase (U / g) ≥7200, and catalase (U / g) ≥1200.

[0024] Further, the corn germ concentrate powder has superoxide dismutase (U / g) ≥11964, glutathione peroxidase (U / g) ≥8202, and catalase (U / g) ≥1366.

[0025] The corn germ concentrate powder can be an antioxidant multi-enzyme prepared by the preparation method.

[0026] In summary, the corn germ concentrate powder has the effect of improving osteoarthritis and can be used to prepare a medicine for treating rheumatoid arthritis. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Effect of the corn germ concentrate powder on the cytotoxicity of TNF-α-induced HFLS-RA.

[0028] In the abscissa, C: not induced by TNF-α; T: induced by TNF-α.

[0029] Figure 2 Effect of the corn germ concentrate powder on the migration ability of TNF-α-induced HFLS-RA.

[0030] A is a migration photo of HFLS-RA cells under different conditions.

[0031] B is the relative migration rate of HFLS-RA cells under different conditions.

[0032] In the abscissa, C: not induced by TNF-α; T: induced by TNF-α.

[0033] Figure 3 Effect of the corn germ concentrate powder on the adhesion ability of TNF-α-induced HFLS-RA.

[0034] In the abscissa, C: not induced by TNF-α; T: induced by TNF-α.

[0035] Figure 4 Effect of the corn germ concentrate powder on the proliferation of hydrogen peroxide-treated MC3T3-E1 cells.

[0036] Figure 5 The effect of corn germ concentrate on the total antioxidant capacity of MC3T3-E1 cells treated with hydrogen peroxide.

[0037] Figure 6 The effect of corn germ concentrate on osteogenic differentiation of MC3T3-E1.

[0038] Figure 7 The effect of corn germ concentrate on the mineralization capacity of MC3T3-E1.

[0039] Figure 8 The effect of corn germ concentrate powder on CAIA model mice with rheumatoid arthritis.

[0040] A shows the swelling of the mouse's right ankle joint.

[0041] B represents the degree of joint swelling in each group of mice.

[0042] Figure 9 The effect of corn germ concentrate on ankle joint pathology in CAIA model mice with rheumatoid arthritis.

[0043] Image A shows an HE staining image of the ankle joint of a mouse in the control group.

[0044] B shows HE staining images of the ankle joints of mice in the model group; Brown arrows: necrotic tissue fragments and inflammatory cells; Green arrow: Severe fibrous connective tissue hyperplasia; Blue arrow: Inflammatory cell infiltration, predominantly granulocytes.

[0045] C shows an HE staining image of the ankle joint of mice in the low-dose group; Brown arrow: Synovial cells detach.

[0046] Image D shows HE staining of the ankle joints of mice in the high-dose group.

[0047] E is an HE staining image of the ankle joint of mice in the positive drug group; Black arrow: Localized synovial cell hyperplasia; Brown arrow: Synovial cell shedding; Yellow arrow: Synovial hemorrhage, vascular congestion.

[0048] Comparison between the model group and the blank group: # p < 0.05, ### p < 0.001, #### p < 0.0001; Comparison between the drug-treated group and the model group: . Detailed Implementation

[0049] Example 1: Preparation of Corn Stew The length of the sprout and the extraction solvent are changed to investigate the influence of the length of the sprout and the extraction solvent on the DPPH scavenging rate in the preparation of the corn milk.

[0050] 100 g of the corn is cleaned and then germinated, 300 mL of the extraction solvent is added to the germinated corn to perform grinding and beating, the grinding is performed for 1 hour to obtain the corn milk.

[0051] The corn milk is left for 30 minutes, the supernatant is filtered, and the DPPH value is measured, and the results are shown in Table 1.

[0052]

[0053] The above results show that when purified water is used as the solvent, the DPPH scavenging rate is obviously improved when the length of the sprout is 4-12 mm, and the DPPH scavenging rate is best when the length of the sprout is 4-8 mm.

[0054] In order to better reflect the overall level and avoid individual errors, the DPPH scavenging rates under the conditions of the same extraction solvent and different lengths of sprouts and the DPPH scavenging rates under the conditions of the same length of sprout and different extraction solvents are investigated, the results are shown in Tables 2 and 3, and according to the results in Tables 2 and 3, it is further determined that the DPPH scavenging rate is better when the length of the sprout is 4-8 mm and the solvent is purified water.

[0055]

[0056] Therefore, in the preparation of the corn milk, the length of the sprout of the corn is selected to be 4-8 mm, and the extraction solvent is purified water.

[0057] Example 2: Preparation of corn germ concentrated powder 10 kg of the corn is cleaned and then germinated, the length of the sprout is 4 mm, 30 L of purified water is added to the germinated corn to perform grinding and beating, the grinding is performed for 3 hours to obtain the corn milk. 3 kg of calcium chloride: kaolin (1:1) is added to the obtained corn milk, stirring is performed for 60 min, the supernatant is taken after standing, 30 g of sodium ascorbate is added, stirring is performed at 60 ℃ for 30 min, 30 g of sodium ascorbate, 20 g of malt dextrin and 10 g of β-cyclodextrin are added, drying is performed at 60 ℃, the powder is crushed, and 3 g of silicon dioxide is added to obtain about 300 g of the corn germ concentrated powder.

[0058] In the prepared corn germ concentrated powder, the superoxide dismutase is 9036 (U / g), the glutathione peroxidase is 6524 (U / g), and the catalase is 835 (U / g).

[0059] Example 3: Influence of different precipitants on enzyme activity in corn germ concentrated powder The kind of the sedimentation agent was changed, and other conditions were the same as in Example 2, the influence of different kinds of the sedimentation agent on the enzyme activity in the corn germ concentrate powder was investigated, and the results are shown in Table 4.

[0060]

[0061] The results in Table 4 show that: when the sedimentation agent is used alone, although the purpose of purification can be achieved, the enzyme activity in the corn germ concentrate powder obtained is lower than that of the corn germ concentrate powder obtained when the ratio of calcium chloride to kaolin is 1:1. It can be seen that when the sedimentation agents are combined, the enzyme activity in the corn germ concentrate powder obtained is higher.

[0062] Example 4 Influence of different combinations of sedimentation agents on enzyme activity in corn germ concentrate powder The ratio of calcium chloride to kaolin was changed, and the influence of different combinations and ratios of kaolin on the enzyme activity in the corn germ concentrate powder was investigated, and the results are shown in Table 5.

[0063]

[0064] The results in Table 5 show that: when calcium chloride is combined with different sedimentation agents, the enzyme activity in the corn germ concentrate powder is different, when calcium chloride is mixed with kaolin, the enzyme activity is improved, and when the ratio of calcium chloride to kaolin is 1:1, the enzyme activity is the highest.

[0065] Example 5 Influence of different stabilizers on the stability of corn germ concentrate powder The corn germ concentrate powder has the effect of scavenging free radicals, which indicates that it has antioxidant property, and thus leads to poor stability. In order to improve the stability, different stabilizers are selected to be protected by wet mixing and drying. Then, the three enzyme activities and the DPPH scavenging rate are measured once a week in a constant temperature oven at 45℃ until the total of the three enzyme activities decreases by 10% (compared with before being put in) and the DPPH scavenging rate decreases by 10% (compared with before being put in). The time used when the total of the three enzyme activities decreases by 10% and the DPPH scavenging rate decreases by 10% is investigated, and the longer the time is, the better the stability is. The results are shown in Table 6.

[0066]

[0067] The results show that: when malt dextrin and β-cyclodextrin are used alone, the time for the total of the three enzyme activities to decrease by 10% is less than 15 weeks, and the time for the DPPH scavenging rate to decrease by 10% is 15-16 weeks, and when the two are combined, the time for the total of the three enzyme activities to decrease by 10% and the DPPH scavenging rate to decrease by 10% can be prolonged, and the time prolonged is different at different ratios, and when the ratio of malt dextrin to β-cyclodextrin is 2:1, the time prolonged is the longest, i.e. the stability is the best. Therefore, the preferred stabilizer in the present application is the combination stabilizer of malt dextrin and β-cyclodextrin in a ratio of 2:1.

[0068] Example 6 Preparation of corn germ concentrate powder Take 10 kg of corn and wash it, then germinate it, with a sprout length of 8 mm. Add 30 L of purified water to the germinated corn and grind it to obtain corn slurry. Add 3 kg of calcium chloride: kaolin (1:1) to the obtained corn slurry, stir for 60 min, stand, take the supernatant, add 30 g of sodium ascorbate, stir at 60°C for 30 min, add 30 g of sodium ascorbate, 20 g of malt dextrin, 10 g of β-cyclodextrin, dry at 60°C, crush, add 3 g of silicon dioxide, and obtain about 300 g of corn germ concentrate powder.

[0069] In the prepared corn germ concentrate powder, the superoxide dismutase is 11964 (U / g), the glutathione peroxidase is 8202 (U / g), and the catalase is 1366 (U / g).

[0070] Example 7 Cytotoxic effect of corn germ concentrate powder on tumor necrosis factor alpha (TNF-α) induced human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) In the synovial tissue of rheumatoid arthritis, fibroblast-like synoviocytes (FLS) are the main effector cells of synovial inflammation and hyperplasia, which play an important role in the pathological process of rheumatoid arthritis. If FLS can be inhibited, it can effectively control synovial inflammation and hyperplasia, and slow down the progression of rheumatoid arthritis. The proliferation, migration and invasion ability of rheumatoid arthritis synoviocytes are greatly enhanced under the stimulation of inflammatory factors (TNF-α, etc.).

[0071] The effect of corn germ concentrate powder (Example 2, hereinafter referred to as concentrate powder) on the proliferation activity of TNF-α induced human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) was investigated by CCK-8 method.

[0072] Logarithmic growth period HFLS-RA was selected, with a cell suspension of 5×10 4 μL per well, 37°C, 5% CO2 incubation for 24 h. The experimental group was added with or without TNF-α with a final concentration of 20 μg / L, 1 h later, different concentrations of concentrate powder (30 μg / mL, 100 μg / mL, 300 μg / mL) were added, the control group was replaced with culture medium containing the same volume of solvent, 3 parallel holes were set for each group, 37°C, 5% CO2 continued to culture for 24 h or 48 h. Discard the supernatant, wash carefully with PBS for 2 times, add 100 μL of freshly prepared culture medium containing 10% CCK8 to each well, incubate at 37°C for 40 min. The optical density value was measured at 450 nm by enzyme marker instrument. The inhibition rate was calculated according to the following formula.

[0073] Survival rate (%) = [1 - (A450 对照组 - A450 给药组 ) / (A450 对照组 - A450 空白 ) ] x 100% The results show that the different concentrations of the concentrated powder all show significant cell proliferation inhibition effect when 20 μg / L TNF-α induces the proliferation of HFLS-RA, the IC50 of 24 h is 124.3 μg / mL, the IC50 of 48 h is 63.1 μg / mL, while in the absence of TNF-α induction, the IC50 of 24 h is 252.1 μg / mL, the IC50 of 48 h is 107.3 μg / mL. The concentrated powder has stronger inhibitory effect on the TNF-α-induced HFLS-RA proliferation Figure 1 ).

[0074] The effect of the corn germ concentrated powder with low enzyme activity on the TNF-α-induced HFLS-RA proliferation activity was investigated by the same method. Among them, the superoxide dismutase in the corn germ concentrated powder is 3589 (U / g), the glutathione peroxidase is 2460 (U / g), and the catalase is 410 (U / g).

[0075] The results show that the corn germ concentrated powder with low enzyme activity has no cell proliferation inhibition effect when acting for 24 h, and the IC50 is 603.8 μg / mL when acting for 48 h, and the inhibitory effect on the TNF-8-induced HFLS-RA cell proliferation is significantly lower than that of the corn germ concentrated powder of Example 2.

[0076] Example 8 Effect of corn germ concentrated powder on TNF-α-induced HFLS-RA migration ability The logarithmic growth period HFLS-RA cells were inoculated in sterile 24-well plates at 1 x 10 5 cells, cultured at 37℃, 5% CO2 for overnight until the cell density reached about 80%, and a "one" was evenly drawn along the diameter of each well under the steel ruler correction. After removing the supernatant, the adherent cells were slowly washed with sterile PBS, and the floating cells were removed. TNF-α with a final concentration of 20 μg / L was added or not added, 1 h later, the concentrated powder (Example 2) was added to an appropriate concentration (10 μg / mL, 30 μg / mL, 100 μg / mL), each group had three duplicate wells, and placed in a cell incubator at 37℃, 5% CO2 for 24 h. Under the inverted microscope, the cell scratch distances of 0 and 24 h were photographed.

[0077] Results showed that the concentrated powder could inhibit cell migration under the action of 20 μg / L TNF-α, and the effect was concentration-dependent, but for HFLS-RA not induced by TNF-α, only 100 μg / mL concentrated powder showed significant migration inhibition effect Figure 2 ).

[0078] Example 9 Effect of corn germ concentrated powder on adhesion ability of TNF-α induced HFLS-RA Add 50 μL of 100 μg / mL Matrigel to the 96-well plate and incubate overnight at 4°C. Add 3×10 5 HFLS cell suspension to the 24-well plate, and after adhesion, add or do not add TNF-α with a final concentration of 20 μg / L, and at the same time, add different concentrations of concentrated powder of Example 3 (10 μg / mL, 30 μg / mL, 100 μg / mL). After 24 h of culture, digest the cells, adjust the cell concentration to 5×10 4 HFLS-RA cells per well to the Matrigel coated 96-well plate; incubate for 1 h, and gently wash with PBS three times to remove unadherent cells; use CCK-8 method to determine the cell stock with TNF-α as the Control group, and calculate the adhesion rate of each group with the cell stock of the blank control group as 100%.

[0079] Results showed that compared with the Control group, the adhesion ability of HFLS-RA cells in the TNF-α group was increased. Compared with the TNF-α group, the 10, 30, and 100 μg / mL concentrated powder groups could significantly reduce the adhesion ability of HFLS-RA cells in a dose-dependent manner Figure 3 ).

[0080] Example 10 Effect of corn germ concentrated powder on proliferation and antioxidant capacity of hydrogen peroxide treated mouse embryonic osteoblasts (MC3T3-E1) After treating MC3T3-E1 cells with hydrogen peroxide, the CCK-8 method was used to investigate the effect of corn germ concentrated powder on the proliferation activity of MC3T3-E1 cells.

[0081] Take the logarithmic phase MC3T3-E1 cells, and adjust the cell concentration to 5×10 3Cells were seeded in 96-well plates at 100 μL / well. After 24 h, the cells were divided into a blank group (C), an oxidative stress injury model group (M, hereinafter referred to as the model group), and a drug-treated group. To establish the oxidative damage model, the MC3T3-E1 cells in the drug-treated group were incubated for 2 h in a growth medium containing 0.3 μmol / L hydrogen peroxide and the drug (concentrated powder concentrations of 30, 100, 300, and 1000 μg / mL as described in Example 2). The control group was incubated for 2 h in a growth medium containing 0.3 μmol / L hydrogen peroxide. Each group had three replicates. The culture medium was then discarded, and the results were measured using the CCK-8 assay.

[0082] The results showed that hydrogen peroxide treatment significantly reduced the cell survival rate in the model group. After administration of concentrated powder, concentrations of 100 μg / mL and above could inhibit hydrogen peroxide-induced MC3T3-E1 cell death and significantly improve the survival rate of MC3T3-E1 cells. Figure 4 ).

[0083] Total antioxidant capacity (T-AOC) assay results showed that 30 μg / mL of concentrated powder could reverse the decrease in antioxidant capacity of MC3T3-E1 cells induced by hydrogen peroxide. After administration of the concentrated powder, the antioxidant capacity of MC3T3-E1 cells was enhanced in a concentration-dependent manner. Figure 5 ).

[0084] Example 11 Effect of corn germ concentrate on osteogenic differentiation of MC3T3-E1 Alkaline phosphatase (ALP) is involved in osteogenic differentiation and is a marker of early osteoblast differentiation; its activity represents the degree of osteogenic differentiation. Log-phase MC3T3-E1 cells were harvested and their concentration adjusted to 1.5 × 10⁻⁶ cells. 4 Cells were seeded in 6-well plates and divided into a control group, a model group (hereinafter referred to as the model group), and a drug-treated group. To establish the oxidative damage model, the control group MC3T3-E1 cells were replaced with growth medium containing 2.5 μmol / L hydrogen peroxide, while the drug-treated group MC3T3-E1 cells were replaced with growth medium containing 2.5 μmol / L hydrogen peroxide and the drug (concentrated powder from Example 3 at concentrations of 30, 100, 300, and 1000 μg / mL), and treated for 2 h. Each group had 3 replicates. Cells were then collected, and alkaline phosphatase activity was calculated according to the ALP assay kit instructions.

[0085] The results show that after hydrogen peroxide treatment, the ALP activity of the model group is significantly reduced, and after giving 100 μg / mL and above concentration of concentrated powder, the ALP activity is significantly increased, and at 300 μg / mL concentration, it reaches the maximum value, and is much higher than the blank group, indicating that the corn germ concentrate powder has a significant ability to promote the osteogenic differentiation of osteoblasts damaged by oxidative stress Figure 6 ).

[0086] Example 12 Effect of corn germ concentrate powder on the mineralization ability of MC3T3-E1 Calcium nodules are the markers of the differentiation and maturation of osteoblasts. β-glycerophosphate, ascorbic acid and low-dose dexamethasone were added to the culture medium to prepare osteogenic induction liquid.

[0087] The cell model was prepared according to the method of Example 8. After the completion of the model, the experimental group was added with drug-containing osteogenic induction liquid (concentrated powder concentration of Example 3 was 30, 100, 300, 1000 μg / mL), and the model group M was added with osteogenic induction liquid, which was replaced once a day, and after 3 weeks, alizarin red staining was used for mineralization nodule staining. The cell culture supernatant was aspirated, and 40 g / L paraformaldehyde was used for fixation for 20 min, and then PBS was used to wash away the paraformaldehyde. Alizarin red staining solution was added for 30 min, and then quickly washed with distilled water for 3 times, and then slightly dried and observed under a microscope. The orange-red precipitate was the mineralized calcium nodule. It can be seen that the mineralized calcium nodule of the model group was significantly reduced, and after being given 100 μg / mL and above concentration of concentrated powder, the mineralized calcium nodule was significantly increased, and at 300 μg / mL concentration, it reached the maximum value, which was equivalent to the blank group, and when the concentration continued to increase, the mineralized calcium nodule decreased, but was still higher than the model group. It indicates that the corn germ concentrate powder has a significant ability to promote the mineralization of osteoblasts damaged by oxidative stress Figure 7 ).

[0088] Example 13 Therapeutic effect of corn germ concentrate powder on CAIA model mice with rheumatoid arthritis The CAIA model is an important animal model for studying rheumatoid arthritis, which is caused by injecting a mixture of monoclonal antibodies of type II collagen and enhancing the immune response of antibodies to joint type II collagen by LPS induction.

[0089] Select 7-week-old female Balb / c mice a total of 30, randomly divided into blank group (Control), model group (Model), concentrated powder high (340 mg / kg) dose group (High), low (170 mg / kg) dose group (Low), positive drug control group (Methotrexate), 6 in each group. 5 days before modeling, the administration group was given Example 3 concentrated powder (twice a day) by gavage, and continuously administered for 16 days. Except for the blank group, the rest of the groups were injected with clone antibody cocktail Arthrogen-ClA 5-Clone Cocktail 150 μL per mouse by tail vein on day 0, and injected with LPS 60 μL per mouse by intraperitoneal injection on day 3. The positive drug group was only given a single dose of methotrexate tablets (2 mg / kg) by gavage on day 0. The joint redness and deformity of the mice were observed daily, the wrist and ankle joint diameters of the mice were measured, and the swelling degree was calculated. Swelling degree = measured diameter - initial diameter, joint swelling degree = sum of limb joint swelling degree. After tail vein injection of antibodies, the model group mice began to show joint redness on day 3, and the degree of joint swelling significantly increased after intraperitoneal injection of LPS for immune enhancement, and the joint redness and deformation were severe on day 10, reaching the peak (P < 0.0001); the joint swelling degree of the methotrexate and concentrated powder high dose group mice was comparable to that of the blank group, and no obvious swelling was observed (Figure 8A). Compared with the model group, the joint swelling degree of the low dose group was significantly reduced (P < 0.01), and the joint swelling degree of the medium and high dose groups was significantly reduced. Figure 8 B) The histopathological results of the ankle joint tissues of the mice in each group showed that the ankle joint tissue structure of the mice in the blank group was clear, the cartilage surface was smooth, the number of chondrocytes was rich, the morphological structure was normal, the arrangement was regular, and no obvious pannus and bone erosion was observed; no obvious hyperplasia was observed, and no obvious inflammatory cell infiltration was observed. Compared with the blank group, the ankle joint tissue structure of the mice in the model group was not clear, and necrotic tissue fragments and inflammatory cells were observed in the joint cavity; the cartilage surface was not smooth, and local cartilage and subchondral bone erosion was observed, and the cartilage and subchondral bone were replaced by hyperplastic connective tissue; the synovial membrane was severely thickened, and severe fibrous connective tissue hyperplasia was observed, accompanied by a large number of inflammatory cell infiltration mainly composed of granulocytes. The pathological features of the ankle joints of the mice in the positive drug group and the low dose group were significantly improved compared with the model group, and local synoviocyte hyperplasia and occasional synoviocyte shedding were observed. The ankle joint structure of the mice in the high dose group showed no obvious abnormalities. It can be seen that the corn germ concentrated powder has obvious bone protection effect on the ankle joints of the CAIA rheumatoid arthritis model mice, and is positively correlated with the dose Figure 9 )。

[0090] In summary, the low, medium and high dose groups of corn germ concentrated powder can significantly reduce the inflammatory response of rheumatoid arthritis, showing different degrees of anti-rheumatoid arthritis effect.

Claims

1. Corn germ concentrate powder, characterized in that, The corn germ concentrate powder contains superoxide dismutase (SOD) ≥ 8500, glutathione peroxidase (GPO) ≥ 5000, and catalase (CAL) ≥ 750, preferably: superoxide dismutase ≥ 9036 U / g, glutathione peroxidase ≥ 6524 U / g, and catalase ≥ 835 U / g.

2. The corn germ concentrate powder according to claim 1, characterized in that, The corn germ concentrate powder contains superoxide dismutase (SOD) ≥ 10000, glutathione peroxidase (GPO) ≥ 7200, and catalase (CAL) ≥ 1200, preferably SOD ≥ 11964, GPO ≥ 8202, and CAL ≥ 1366.

3. The corn germ concentrate powder according to claim 1 or 2, characterized in that, After washing the corn, germinate it. Add water to the germinated corn and grind it into a pulp. Add a settling agent to the corn pulp and let it stand. Take the supernatant, add some sodium ascorbate and concentrate it under controlled temperature. Add the remaining sodium ascorbate and stabilizer to the concentrate, dry it under controlled temperature, pulverize it, and mix in an appropriate amount of silica to obtain the final product. The sprouted buds are 4-12 mm long; The grinding time is 1-3 hours; The sodium ascorbate contained in the corn germ concentrate is 15-25%; The settling agent is a mixture of calcium chloride and kaolin, with a mass ratio of calcium chloride to kaolin of 1:0.5-1.5; The mass ratio of corn to settling agent is 10:2.5-3.5; The stabilizer is a mixture of maltodextrin and β-cyclodextrin, wherein the mass ratio of maltodextrin to β-cyclodextrin is 2-3:1-2; The stabilizer constitutes 8-12% of the corn germ concentrate by mass. The temperature for temperature-controlled concentration and temperature-controlled drying is 60-70℃, and the temperature-controlled concentration time is 25-35 minutes.

4. The corn germ concentrate powder according to claim 1 or 2, characterized in that, After washing the corn, germinate it. Add 3-5 times the amount of water to the corn that has germinated to 4-8mm and grind it for 3 hours. Add a settling agent to the corn pulp and let it stand. Take the supernatant, add some sodium ascorbate and concentrate it under controlled temperature. Add the remaining sodium ascorbate and stabilizer to the concentrate, dry it under controlled temperature, pulverize it, and mix in an appropriate amount of silica to obtain the final product. The settling agent is a mixture of calcium chloride and kaolin, with a mass ratio of calcium chloride to kaolin of 1:

1. The mass ratio of corn to flocculant is 10:3; The stabilizer is a mixture of maltodextrin and β-cyclodextrin, wherein the mass ratio of maltodextrin to β-cyclodextrin is 2:

1. The sodium ascorbate contained in the corn germ concentrate accounted for 20% of the total concentration. The mass ratio of the first added sodium ascorbate to the remaining sodium ascorbate is 1:1; The stabilizer accounts for 10% of the mass of the corn germ concentrate.

5. The use of the corn germ concentrate powder according to claim 1 or 2 in the preparation of a medicament for improving osteoarthritis.

6. The use of the corn germ concentrate powder according to claim 1 or 2 in the preparation of a medicament for treating rheumatoid arthritis.

7. The application according to claim 6, characterized in that, The rheumatoid arthritis mentioned is rheumatoid arthritis caused by stimulation of tumor necrosis factor α.

8. The use of the corn germ concentrate powder according to claim 1 or 2 in the preparation of food that helps improve bone density.

9. A method for preparing corn germ concentrate powder, characterized in that, After washing the corn, germinate it. Add 3-5 times the amount of water to the corn that has germinated to 4-8mm and grind it into a pulp. Add a settling agent to the corn pulp and let it stand. Take the supernatant, add some sodium ascorbate and concentrate it under controlled temperature. Add the remaining sodium ascorbate and stabilizer to the concentrate, dry it under controlled temperature, pulverize it, and mix in an appropriate amount of silica to obtain the final product. The settling agent is a mixture of calcium chloride and kaolin, with a mass ratio of calcium chloride to kaolin of 1:0.5-1.5; The mass ratio of corn to settling agent is 10:2.5-3.5; The stabilizer is a mixture of maltodextrin and β-cyclodextrin, wherein the mass ratio of maltodextrin to β-cyclodextrin is 2-3:1-2; The stabilizer constitutes 8-12% of the corn germ concentrate by mass. The sodium ascorbate contained in the corn germ concentrate is 15-25%.

10. The method for preparing corn germ concentrate powder according to claim 9, characterized in that, After washing the corn, germinate it. Add 3-5 times the amount of water to the corn that has germinated to 4-8mm and grind it for 3 hours. Add a settling agent to the corn pulp and let it stand. Take the supernatant, add some sodium ascorbate and concentrate it under controlled temperature. Add the remaining sodium ascorbate and stabilizer to the concentrate, dry it under controlled temperature, pulverize it, and mix in an appropriate amount of silica to obtain the final product. The settling agent is a mixture of calcium chloride and kaolin, with a mass ratio of calcium chloride to kaolin of 1:

1. The mass ratio of corn to flocculant is 10:3; The stabilizer is a mixture of maltodextrin and β-cyclodextrin, wherein the mass ratio of maltodextrin to β-cyclodextrin is 2:

1. The sodium ascorbate contained in the corn germ concentrate accounted for 20% of the total concentration. The mass ratio of the first added sodium ascorbate to the remaining sodium ascorbate is 1:1; The stabilizer accounts for 10% of the mass of the corn germ concentrate.

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