A nutritional composition for improving bone density and use thereof

Abstract

The present application relates to the field of food technology, and specifically discloses a nutritional composition for improving bone density and application thereof. The present application provides a nutritional composition for improving bone density prepared from pueraria lobata, colostrum basic protein and a selenium source. The nutritional composition can significantly up-regulate the expression of osteogenesis marker genes such as ALP, RUNX2, Colla1, OCN and OPG in osteoblasts, down-regulate the expression of RANKL gene, and regulate the OPG / RANKL ratio to tend to be balanced, while reducing the level of inflammatory factors. The nutritional composition synergistically regulates bone metabolism through the triple action mechanisms of promoting bone formation, inhibiting bone resorption and anti-inflammation, effectively increases bone density, and realizes the prevention and intervention treatment of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of food technology, and specifically discloses a nutritional composition for improving bone density and its application. Background Technology

[0002] Osteoporosis is a major public health challenge worldwide, with a high clinical incidence in postmenopausal women and the elderly. Pregnancy and lactation-associated osteoporosis (PLO) refers to osteoporosis diagnosed in late pregnancy to 18 months postpartum, especially in the postpartum or early lactation period. It is characterized by low bone mass and bone microarchitectural deterioration, often presenting as severe lower back pain or mild hip pain, and is easily overlooked clinically. Studies have shown that the median bone mineral density loss in the lumbar spine of breastfeeding mothers within 6 months postpartum is 6%-8%.

[0003] Current conventional treatments for osteoporosis mostly rely on simple calcium supplementation, combined with vitamin D supplementation, or one-way intervention using anti-inflammatory drugs and bone resorption inhibitors. These approaches have limited pathways of action and cannot simultaneously address the three regulatory needs of promoting new bone formation, blocking bone loss, and alleviating local bone inflammation. For individuals with specific physiological conditions, a significant decrease in bone density poses multiple health risks: continuous bone loss directly damages the internal microstructure of bones, reducing bone strength and resilience, significantly increasing the incidence of vertebral compression fractures, hip fractures, and rib fractures. It can also induce persistent, severe back and hip pain, severely impacting mobility and daily life. Furthermore, the damaged bone microstructure is difficult to repair itself with a single intervention. Conventional single-nutrient supplementation or chemical drug treatment modalities are not ideal for improving overall bone density or treating osteoporosis. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a nutritional composition for improving bone density and its applications. This invention combines kudzu root, colostrum basic protein, and a selenium source to prepare a nutritional composition that exhibits a significant synergistic effect in increasing bone density and alleviating osteoporosis. Based on these advantages, the nutritional composition for improving bone density provided by this invention can be widely used in areas such as improving bone density and preventing osteoporosis.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a nutritional composition for improving bone density, comprising kudzu root, colostrum basic protein, and a selenium source.

[0006] This invention combines kudzu root, colostrum basic protein, and selenium source to prepare a nutritional composition for improving bone density. The three raw materials in this composition can produce significant synergistic effects in improving osteoblast survival rate and inducing the expression of osteogenic marker genes. It can systematically regulate the body's bone metabolism homeostasis, effectively improve bone density, and alleviate osteoporosis-related symptoms.

[0007] In this invention, the selenium source refers to various raw materials that can provide selenium (Se).

[0008] For example, the selenium source includes at least one of sodium selenite, selenoprotein, selenium-enriched yeast, selenized carrageenan, or L-seleno-methylselenocysteine.

[0009] Preferably, the mass ratio of kudzu root, colostrum basic protein and selenium source is (200-1600):(50-200):(1-5); wherein the mass of selenium source is expressed as Se.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned nutritional composition for improving bone density, the method comprising the following steps: S1. Mix kudzu root with water and gelatinize to obtain a gelatinized dispersion; S2. Add cellulase to the gelatinized dispersion, perform enzymatic hydrolysis, and then subject it to intermittent ultrasonic treatment to obtain an ultrasonically treated solution; S3. Add colostrum basic protein and selenium source to the ultrasonic treatment solution to obtain the nutritional composition for improving bone density.

[0011] This invention involves gelatinizing kudzu root and then subjecting it to a combination of enzymatic hydrolysis and intermittent ultrasonic treatment to release active ingredients, including puerarin. The gelatinized kudzu root is then blended with colostrum basic protein. The addition of a selenium source allows selenium atoms to form selenium-sulfur covalent bonds with the thiol groups of the colostrum basic protein, while simultaneously forming hydrogen bonds with the hydroxyl groups in the gelatinized starch of the kudzu root. This process self-assembles the kudzu root into a stable ternary complex, resulting in a nutritional composition that improves bone density.

[0012] Preferably, the mass ratio of kudzu root to water is 1:8-12. The kudzu root is kudzu root powder that has passed through a 60-100 mesh sieve.

[0013] Preferably, the gelatinization temperature is 85℃-95℃, and the gelatinization time is 20min-30min.

[0014] Preferably, step S2 specifically includes the following steps: passing the gelatinized dispersion through a 100-300 mesh sieve to obtain a filtrate; adjusting the pH of the filtrate to 4.5-5.5 and the temperature to 45℃-55℃; adding cellulase; incubating and hydrolyzing for 0.5h-2h to obtain an enzymatic hydrolysate; inactivating the enzyme in the enzymatic hydrolysate; cooling to 45℃-50℃; and performing intermittent ultrasonic treatment to obtain an ultrasonically treated solution.

[0015] Preferably, the power of the intermittent ultrasonic treatment is 200W-300W and the frequency is 20kHz-40kHz.

[0016] Preferably, the intermittent ultrasound treatment mode is as follows: each ultrasound treatment lasts 4-6 minutes, followed by a 1.5-2.5 minute pause.

[0017] Preferably, the total processing time for the intermittent ultrasound is 25-45 minutes.

[0018] Preferably, step S3 specifically includes the following steps: adding colostrum basic protein to the ultrasonic treatment solution, mixing evenly, adding selenium source, and then adding ascorbic acid to obtain the nutritional composition for improving bone density.

[0019] For example, using sodium selenite as the selenium source, the mass ratio of ascorbic acid to sodium selenite is 3.5-4.5:1.

[0020] For example, after adding ascorbic acid, the operation should be carried out in the dark, and the stirring temperature should be controlled at 40℃-45℃ to ensure the reduction and complexation effect.

[0021] In this invention, sodium selenite is used as an example of the added selenium source. Ascorbic acid will neutralize the selenium ions (Se) in sodium selenite. 4+ The selenium atoms are reduced to selenium atoms; the selenium atoms form selenium-sulfur covalent bonds with the thiol groups of colostrum basic proteins, and at the same time form hydrogen bonds with the hydroxyl groups of gelatinized starch in kudzu root, self-assembling to form a stable ternary complex.

[0022] Thirdly, the present invention provides the application of the nutritional composition for improving bone density prepared by the method of preparing the nutritional composition for improving bone density in the first aspect or the method of preparing the nutritional composition for improving bone density in the second aspect in the preparation of products for improving bone density.

[0023] For example, the product for improving bone density includes at least one of ordinary foods (such as functional foods) or health foods.

[0024] Fourthly, the present invention provides the use of the nutritional composition for improving bone density prepared by the method of preparing the nutritional composition for improving bone density in the first aspect or the method of preparing the nutritional composition for improving bone density in the second aspect in the preparation of products for preventing or treating osteoporosis.

[0025] For example, the osteoporosis includes osteoporosis during pregnancy and lactation.

[0026] Fifthly, the present invention provides a product for improving bone density, the product comprising the nutritional composition for improving bone density described in the first aspect.

[0027] In a sixth aspect, the present invention provides a product for preventing and treating osteoporosis, the product comprising the nutritional composition for improving bone density described in the first aspect.

[0028] For example, the nutritional composition for improving bone density can be used to prepare formula milk powder, liquid milk or nutritional supplements suitable for pregnant and postpartum women, so as to improve the bone density of pregnant and postpartum women and prevent osteoporosis.

[0029] This invention provides a nutritional composition for improving bone mineral density, derived from kudzu root, colostrum basic protein, and selenium source. This composition significantly upregulates the expression of osteogenic marker genes such as ALP, RUNX2, Colla1, OCN, and OPG in osteoblasts, downregulates RANKL gene expression, and balances the OPG / RANKL ratio, while simultaneously reducing inflammatory factor levels. This nutritional composition effectively increases bone mineral density through a triple mechanism of action—promoting bone formation, inhibiting bone resorption, and reducing inflammation—synergistically regulating bone metabolism and achieving prevention and intervention for osteoporosis. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a statistical graph showing the cell survival rate in different groups in Example 1 of the present invention; Figure 2 This is a statistical graph showing the relative gene expression levels of osteogenic markers ALP, RANKL, RUNX2, Colla1, OCN, and OPG in different groups in Example 2 of the present invention. Figure 3 This is a statistical graph showing the relative gene expression levels of inflammatory factors IL-6, IL-1β, IL-12, and INOS in different groups in Example 2 of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] The colostrum basic protein, sodium selenite, kudzu root powder and cellulase used in this invention are all commercially available products. The colostrum basic protein raw material contains 98% colostrum basic protein; the sodium selenite raw material contains 0.5% selenium (calculated as Se). The raw material for kudzu root powder is dried kudzu root produced in Shaoguan, Guangdong Province. It is prepared by crushing and passing through an 80-mesh sieve. According to the test, the kudzu root powder selected in this invention contains 8.0% puerarin and the enzyme activity of cellulase is 50,000 U / g.

[0034] Example 1 This invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 400g of kudzu root powder, 100g of colostrum basic protein raw material, and 2g of sodium selenite raw material (calculated as Se).

[0035] This invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: S1. Mix kudzu root powder that has passed through an 80-mesh sieve with purified water at a mass ratio of 1:10, heat to 85℃ and gelatinize for 30 minutes to form a gelatinized dispersion; S2. The gelatinized dispersion is passed through a 200-mesh sieve to obtain a filtrate. The pH of the filtrate is adjusted to 5.0 and the temperature is set to 50°C. Cellulase with an enzyme activity of 50,000 U / g and a mass of 1.0% of the filtrate is added. The mixture is incubated for 1 hour to obtain an enzymatic hydrolysate. The enzyme hydrolysate was inactivated at 90°C for 10 min, then cooled to 45°C and subjected to intermittent ultrasonic treatment for 42 min to obtain an ultrasonically treated solution.

[0036] The intermittent ultrasound treatment has a power of 250W and a frequency of 30kHz. The working mode of the intermittent ultrasound treatment is: 5 minutes of ultrasound treatment followed by 2 minutes of ultrasound cessation; 6 cycles are performed.

[0037] S3. Add food-grade sodium bicarbonate to the ultrasonic treatment solution to adjust the pH of the system to 6.5, then add colostrum basic protein raw material and stir for 20 min; then add sodium selenite and continue stirring for 15 min to ensure that the selenite ions are fully in contact with the colostrum basic protein in the dispersion; finally add ascorbic acid in an amount 4 times the mass of sodium selenite, and stir in the dark throughout the process, controlling the temperature at 40℃, and continue stirring for 30 min to obtain the kudzu root-CBP-selenium composite solution; The kudzu-CBP-selenium composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain nutritional composition I for improving bone density.

[0038] Example 2 This invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 800g of kudzu root powder, 100g of colostrum basic protein raw material, and 2g of sodium selenite raw material (calculated as Se).

[0039] The method for preparing the nutritional composition for improving bone density in this embodiment of the invention is the same as that in Example 1, and the nutritional composition II for improving bone density is finally obtained.

[0040] Example 3 This invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 1600g of kudzu root powder, 100g of colostrum basic protein raw material, and 2g of sodium selenite raw material (calculated as Se).

[0041] The method for preparing the nutritional composition for improving bone density in this embodiment of the invention is the same as that in Example 1, and the nutritional composition III for improving bone density is finally obtained.

[0042] Example 4 This invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 400g of kudzu root powder, 50g of colostrum basic protein raw material, and 2g of sodium selenite raw material (calculated as Se).

[0043] This invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: S1. Mix kudzu root powder that has passed through an 80-mesh sieve with purified water at a mass ratio of 1:8, heat to 90℃ and gelatinize for 20 minutes to form a gelatinized dispersion; S2. The gelatinized dispersion is passed through a 100-mesh sieve to obtain a filtrate. The pH of the filtrate is adjusted to 4.5 and the temperature is set to 55°C. Cellulase with an enzyme activity of 50,000 U / g and a mass of 1.0% of the filtrate is added. The mixture is incubated for 0.5 hours to obtain an enzymatic hydrolysate. The enzyme hydrolysate was inactivated at 90°C for 10 min, then cooled to 50°C and subjected to intermittent ultrasonic treatment for 27.5 min to obtain the ultrasonically treated solution.

[0044] The intermittent ultrasound treatment has a power of 300W and a frequency of 40kHz. The working mode of the intermittent ultrasound treatment is: 4 minutes of ultrasound treatment followed by 1.5 minutes of ultrasound cessation; 5 cycles are performed.

[0045] S3. Add food-grade sodium bicarbonate to the ultrasonic treatment solution to adjust the pH of the system to 6.5, then add colostrum basic protein raw material and stir for 15 min; then add sodium selenite and continue stirring for 15 min to ensure that the selenite ions are fully in contact with the colostrum basic protein in the dispersion; finally add ascorbic acid in an amount 4 times the mass of sodium selenite, and stir in the dark throughout the process, controlling the temperature at 45℃, and continue stirring for 30 min to obtain the kudzu root-CBP-selenium composite solution; The kudzu-CBP-selenium composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain the nutritional composition IV for improving bone density.

[0046] Example 5 This invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 1600g of kudzu root powder, 200g of colostrum basic protein raw material, and 5g of sodium selenite raw material (calculated as Se).

[0047] This invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: S1. Mix kudzu root powder that has passed through an 80-mesh sieve with purified water at a mass ratio of 1:12, heat to 90℃ and gelatinize for 25 minutes to form a gelatinized dispersion; S2. The gelatinized dispersion is passed through a 300-mesh sieve to obtain a filtrate. The pH of the filtrate is adjusted to 4.5 and the temperature is set to 45°C. Cellulase with an enzyme activity of 50,000 U / g and a mass of 1.0% of the filtrate is added. The mixture is incubated for 2 hours to obtain an enzymatic hydrolysate. The enzyme hydrolysate was inactivated at 90°C for 10 min, then cooled to 50°C and subjected to intermittent ultrasonic treatment for 42.5 min to obtain an ultrasonically treated solution.

[0048] The intermittent ultrasound treatment has a power of 200W and a frequency of 20kHz. The working mode of the intermittent ultrasound treatment is: 6 minutes of ultrasound treatment followed by a 2.5-minute pause; 5 cycles are performed.

[0049] S3. Add food-grade sodium bicarbonate to the ultrasonic treatment solution to adjust the pH of the system to 6.5, then add colostrum basic protein raw material and stir for 20 min; then add sodium selenite raw material and continue stirring for 20 min to ensure that the selenite ions are fully in contact with the colostrum basic protein in the dispersion; finally add ascorbic acid in an amount 4 times the mass of sodium selenite, and stir in the dark throughout the process, controlling the temperature at 40℃, and continue stirring for 30 min to obtain the kudzu root-CBP-selenium composite solution; The kudzu-CBP-selenium composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain nutritional composition V for improving bone density.

[0050] Comparative Example 1 This invention provides a comparative example of a nutritional composition for improving bone density. The raw materials and amounts used to prepare this nutritional composition are the same as in Example 2. The preparation method is basically the same as in Example 2, except that in step S2 of Example 2, "the enzyme hydrolysate is inactivated at 90°C for 10 minutes, then cooled to 45°C, and subjected to intermittent ultrasonic treatment for 42 minutes to obtain an ultrasonic treatment solution. The power of the intermittent ultrasonic treatment is 250W, and the frequency is 30kHz; the working mode of the intermittent ultrasonic treatment is: every 5 minutes of ultrasonic treatment, stop for 2 minutes; perform 6 cycles," is replaced with "the enzyme hydrolysate is inactivated at 90°C for 10 minutes, then cooled to 45°C, and subjected to continuous ultrasonic treatment for 30 minutes to obtain an ultrasonic treatment solution. The power of the ultrasonic treatment is 250W, and the frequency is 30kHz." The remaining steps and parameters are the same as in Example 2, ultimately yielding the nutritional composition for improving bone density, Part I.

[0051] Comparative Example 2 This invention provides a comparative example of a nutritional composition for improving bone density. The raw materials and amounts used to prepare this nutritional composition are the same as in Example 2. The preparation method is basically the same as in Example 2, except that step S2 of Example 2 is replaced with: "Passing the gelatinized dispersion through a 200-mesh sieve to obtain a filtrate, subjecting the filtrate to continuous ultrasonic treatment for 30 minutes to obtain an ultrasonically treated solution; wherein the ultrasonic treatment power is 250W and the frequency is 30kHz; adjusting the pH of the ultrasonically treated solution to 5.0 and the temperature to 50℃, adding cellulase with an enzyme activity of 50000U / g and 1.0% of the filtrate mass, incubating for enzymatic hydrolysis for 1 hour to obtain an enzymatic hydrolysate; inactivating the enzyme in the enzymatic hydrolysate at 90℃ for 10 minutes, and then cooling to 45℃ to obtain an enzyme-inactivated hydrolysate"; replacing "ultrasonically treated solution" in step S3 of Example 2 with "enzyme-inactivated hydrolysate"; the remaining steps and parameters are the same as in Example 2, ultimately yielding the nutritional composition for improving bone density, Part II.

[0052] Comparative Example 3 The present invention provides a nutritional composition for improving bone density in a comparative example. The raw materials and amounts used to prepare the nutritional composition are the same as those in Example 2.

[0053] The comparative example of this invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: S1. Mix kudzu root powder that has passed through an 80-mesh sieve with purified water at a mass ratio of 1:10, heat to 85℃ and gelatinize for 30 minutes to form a gelatinized dispersion; S2. The gelatinized dispersion is passed through a 200-mesh sieve to obtain a filtrate. Colostrum basic protein raw material powder is added to the filtrate and stirred for 20 minutes. Then sodium selenite raw material powder is added and stirred for another 15 minutes to ensure that the selenite ions are fully in contact with the colostrum basic protein in the dispersion. Finally, ascorbic acid in an amount four times the mass of sodium selenite is added. The mixture is stirred in the dark throughout the process, and the temperature is controlled at 40-45°C. The mixture is stirred for another 30 minutes to obtain a kudzu-CBP-selenium composite solution. The kudzu-CBP-selenium composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain the nutritional composition for improving bone density, Pair III.

[0054] Comparative Example 4 The present invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 800g of kudzu root powder and 100g of colostrum basic protein raw material.

[0055] The comparative example of this invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: Steps S1 and S2 are the same as those in Example 2 in terms of operation and parameters, and an ultrasonic treatment solution is obtained.

[0056] S3. Add food-grade sodium bicarbonate to the ultrasonic treatment solution to adjust the pH of the system to 6.5, then add colostrum basic protein raw material, stir for 20 min, and obtain kudzu root-CBP composite solution; The kudzu-CBP composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain the nutritional composition for improving bone density.

[0057] Comparative Example 5 The present invention provides a nutritional composition for improving bone density, comprising the following raw materials by weight: 100g of colostrum basic protein raw material and 2g of sodium selenite raw material (calculated as Se).

[0058] The comparative example of this invention also provides a method for preparing the above-mentioned nutritional composition for improving bone density, specifically including the following steps: A 0.2% colostrum basic protein solution was prepared. The colostrum alkaline protein solution was stirred with sodium selenite raw material for 15 minutes, and then ascorbic acid in 4 times the mass of sodium selenite was added. The stirring was carried out in the dark and the temperature was controlled at 40℃. The stirring was continued for 30 minutes to obtain CBP-selenium complex solution. The CBP-selenium composite solution was dialyzed with purified water as the dialysis medium and replaced every 6 hours (the molecular weight cutoff of the dialysis bag was 3 kDa). After dialysis 4 times, the dialysate was collected and concentrated under reduced pressure at 45°C and a vacuum degree ≤ -0.08 MPa until the solid content was 15%~20%. Then it was freeze-dried to obtain the nutritional composition for improving bone density (P<0.5).

[0059] Comparative Example 6 This invention provides a comparative example of kudzu root freeze-dried powder, the preparation method of which includes the following steps: S1. Mix kudzu root powder that has passed through an 80-mesh sieve with purified water at a mass ratio of 1:10, heat to 85℃ and gelatinize for 30 minutes to form a gelatinized dispersion; S2. The gelatinized dispersion is passed through a 200-mesh sieve to obtain a filtrate. The pH of the filtrate is adjusted to 5.0 and the temperature is set to 50°C. Cellulase with an enzyme activity of 50,000 U / g and a mass of 1.0% of the filtrate is added. The mixture is incubated for 1 hour to obtain an enzymatic hydrolysate. The enzyme hydrolysate was inactivated at 90°C for 10 min, then cooled to 45°C and subjected to intermittent ultrasonic treatment for 42 min to obtain an ultrasonically treated solution.

[0060] The intermittent ultrasound treatment has a power of 250W and a frequency of 30kHz. The working mode of the intermittent ultrasound treatment is: 5 minutes of ultrasound treatment followed by 2 minutes of ultrasound cessation; 6 cycles are performed.

[0061] S3. Add food-grade sodium bicarbonate to the ultrasonic treatment solution to adjust the pH of the system to 6.5. Concentrate under reduced pressure at 45℃ and vacuum degree ≤ -0.08MPa until the solid content is 15%~20%. Freeze dry to obtain kudzu root freeze-dried powder.

[0062] Comparative Example 7 This invention provides a selenium raw material in a comparative example, the preparation method of which includes the following steps: A 0.5% sodium selenite solution was prepared, and ascorbic acid in an amount four times the mass of sodium selenite was added. The mixture was stirred in the dark, and the temperature was controlled at 40-45°C. The mixture was stirred for another 30 minutes and then freeze-dried to obtain the selenium raw material.

[0063] Example of effect 1 This invention uses the nutritional compositions for improving bone density prepared in Examples 1-3 as examples, and compares them with the nutritional compositions for improving bone density prepared in Comparative Examples 1-5, and the kudzu root freeze-dried powder and selenium raw material prepared in Comparative Examples 6-7, to investigate the effects of kudzu root, colostrum basic protein, and selenium source as a single component or in pairs, as well as the preparation method, on the survival rate of MC3T3-E1 osteoblasts. The specific details are as follows: MC-3T3E1 osteoblasts, monolayered and adherent in DMEM basal medium containing 10% fetal bovine serum, were passaged in a 37°C, 5% CO2 incubator until the logarithmic growth phase. These logarithmic-phase osteoblasts were then added to 96-well plates and randomly divided into 16 groups. After overnight culture, different final concentrations of a bone mineral density-enhancing nutrient composition, lyophilized kudzu root powder, or selenium feedstock, and / or dexamethasone were added to each well according to the group. The plates were then incubated at 37°C, 5% CO2 for 24 hours. Then, 10 μL of CCK-8 reaction solution was added, and incubation continued for 1-2 hours. The absorbance (A) value at 450 nm was measured using a microplate reader. Cell viability was calculated based on the A value. It should be noted that negative control groups 1 and 2 did not contain osteoblasts. Unless otherwise specified, "DMEM basal medium containing 10% fetal bovine serum" in the following groups is abbreviated as "medium"; the experimental group containing osteoblasts contains 100 μL of DMEM basal medium with a cell concentration of 5 × 10⁻⁶ cells / mL. 4 osteoblast culture medium (obtained from the above culture medium) with cells / mL.

[0064] The specific grouping arrangements are as follows: Normal group: osteoblasts; Negative control group 1: Contains only culture medium; Negative control group 2: culture medium and a nutritional composition for improving bone density, kudzu root freeze-dried powder or selenium raw material at the same concentration as the experimental group; Negative control group 3: osteoblasts and a nutritional composition for improving bone density, kudzu root freeze-dried powder or selenium raw material at the same concentration as the experimental group; Model group: osteoblasts were treated with a final concentration of 10... -5 mol / L dexamethasone; Example 1 group: osteoblasts were added with a final concentration of 10 -5 The intervention involved administering dexamethasone at a concentration of mol / L, along with a bone density-improving nutritional composition I. The nutritional composition solution was prepared as follows: one-twentieth of the total mass of the bone density-improving nutritional composition I was added to 100 mL of purified water and mixed thoroughly. This mixture was then diluted 20 times to obtain the intervention nutritional composition solution. Based on the original feed ratios of the raw materials, the final concentrations of each raw material in this intervention system (calculated as original feed ratios) were: 4 mg / mL kudzu root powder, 1 mg / mL colostrum basic protein (CBP), and 20 μg / mL sodium selenite (as Se). Example 2 group: osteoblasts were added with a final concentration of 10 -5 The intervention involved administering dexamethasone at a concentration of mol / L, along with a bone density-improving nutritional composition II. The nutritional composition solution was prepared as follows: one-twentieth of the total mass of the bone density-improving nutritional composition II was added to 100 mL of purified water and mixed thoroughly, then diluted 20 times to obtain the intervention nutritional composition solution. Based on the original feed ratios of the raw materials, the final concentrations of each raw material in this intervention system (based on the original feed ratios) were: 8 mg / mL kudzu root powder, 1 mg / mL colostrum basic protein (CBP), and 20 μg / mL sodium selenite (as Se). Example 3 group: osteoblasts were added with a final concentration of 10 -5 The intervention involved administering dexamethasone at a concentration of mol / L, along with a bone mineral density-enhancing nutritional composition III. The nutritional composition solution was prepared as follows: one-twentieth of the total mass of the bone mineral density-enhancing nutritional composition III was added to 100 mL of purified water and mixed thoroughly. This mixture was then diluted 20 times to obtain the intervention nutritional composition solution. Based on the original feed ratios of the raw materials, the final concentrations of each raw material in this intervention system (based on the original feed ratios) were: 8 mg / mL kudzu root powder, 1 mg / mL colostrum basic protein (CBP), and 20 μg / mL sodium selenite (as Se). Comparative Example 1: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at mol / L was added to a nutrient composition for improving bone density to intervene in group I. The preparation method of the nutrient composition solution and the final concentration of each raw material in the intervention system (based on the original feed) were the same as those in group 2 of Example 2; Comparative Example 2: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at mol / L was added to the intervention for group II, along with a nutritional composition for improving bone density. The preparation method of the nutritional composition solution and the final concentration (based on the original feed) of each raw material in the intervention system were the same as those in group 2 of Example 2. Comparative Example 3: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at mol / L was added to a nutrient composition for improving bone density to intervene in group III. The preparation method of the nutrient composition solution and the final concentration of each raw material in the intervention system (based on the original feed) were the same as those in group 2 of Example 2. Comparative Example 4: Osteoblasts were treated with a final concentration of 10... -5Dexamethasone at mol / L was added to a nutritional composition for improving bone density to intervene in group IV. The nutritional composition solution was prepared in the same way as in Example 2. Based on the original feed ratio of each raw material in the composition, the final concentration of each raw material in this intervention system (based on the original feed ratio) was: 8 mg / mL of kudzu root powder and 1 mg / mL of colostrum basic protein (CBP).

[0065] Comparative Example 5: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at mol / L was added to the intervention for group V, along with a nutritional composition for improving bone density. The preparation method of the nutritional composition solution was the same as that of group 2 in Example. Based on the original feed ratio of each raw material in the composition, the final concentration of each raw material in this intervention system (based on the original feed ratio) was: colostrum basic protein (CBP) 1 mg / mL and sodium selenite (as Se) 20 μg / mL.

[0066] Comparative Example 6: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at a concentration of mol / L was added to lyophilized kudzu root powder for intervention. The preparation method of the lyophilized kudzu root powder solution was the same as that in Example 2. Based on the original feed ratio of kudzu root in the lyophilized kudzu root powder, the final concentration of kudzu root in this intervention system (based on the original feed ratio) was 8 mg / mL of kudzu root powder.

[0067] Comparative Example 7: Osteoblasts were treated with a final concentration of 10... -5 Dexamethasone at a concentration of mol / L was added simultaneously with the selenium feedstock prepared in Comparative Example 7 for intervention. The preparation method of the selenium feedstock solution was the same as that of Example 2. Based on the original feed ratio of selenium in the selenium feedstock, the final concentration of selenium in this intervention system (based on the original feed ratio) was 20 μg / mL.

[0068] The formula for calculating cell viability is as follows: Cell viability (%) = (A 实验组 -A 空白组 ) / (A 对照组 -A 空白组 ) × 100%.

[0069] When calculating the cell viability of the remaining 11 groups (excluding the model group and negative control groups 1-3), the blank group in the formula refers to negative control group 2, and the control group refers to negative control group 3. When calculating the cell viability of the model group, the blank group in the formula refers to negative control group 1, and the control group refers to the normal group.

[0070] The statistical results of cell viability in different groups are shown in Table 1. The statistical graph of cell viability in different groups is shown below. Figure 1 As shown.

[0071] Table 1 Note: Different letter subscripts after the data in the same column indicate significant differences (P<0.05).

[0072] Depend on Figure 1 As shown in Table 1, the nutritional compositions for improving bone density prepared by the methods provided in Examples 1-3 of this invention significantly enhance the survival rate of dexamethasone-treated osteoblasts. Compared with the results of Example 1 and Comparative Examples 1-3, the intermittent hydrolysis of kudzu root powder after enzymatic hydrolysis significantly improved the survival rate of dexamethasone-treated osteoblasts. The combination of kudzu root powder, selenium source, or colostrum basic protein, or any single component, did not promote the survival rate of osteoblasts.

[0073] Example 2 This invention investigates the effects of a nutritional composition for improving bone mineral density on osteogenic markers and inflammatory factors in osteoblasts MC3T3-E1. The specific details are as follows: Logarithmic-phase osteoblasts MC3T3-E1 were cultured in 6-well plates with a final concentration of 10... -5 Osteoporosis cell models were constructed by treating cells with mol / L dexamethasone solution for 15 h and randomly dividing them into 12 groups. Following the classification method and treatment dosage in Example 1, the cells were further divided into a model group, Examples 1-3 groups, and Comparative Examples 1-7 groups. Osteoblasts were treated with the corresponding components and dosages for 24 h. RNA was then extracted according to the kit requirements, and the relative expression levels of osteogenic specific markers and inflammatory factors were detected by qPCR. The osteogenic markers selected in this invention include alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), osteogenic-associated transcription factor-2 (RUNX2), type I collagen (Colla1), and osteocalcin (OCN), with the gene encoding β-actin used as an internal reference gene. Inflammatory factors included IL-6, IL-1β, IL-12, and INOS.

[0074] The specific primer sequences for osteogenic markers and inflammatory factors are shown in Table 2.

[0075] Table 2 The qPCR reaction conditions were as follows: pre-denaturation at 95℃ for 10 min; followed by 40 cycles, each cycle consisting of: denaturation at 95℃ for 15 s, annealing at 60℃ for 30 s, extension at 72℃ for 10 s; and final extension at 72℃ for 10 min.

[0076] The statistical results of gene expression levels of osteogenic markers ALP, RANKL, RUNX2, Colla1, OCN, and OPG in different groups are shown in Table 3. The statistical graph of the relative gene expression levels of osteogenic markers ALP, RANKL, RUNX2, Colla1, OCN, and OPG in different groups is shown below. Figure 2 As shown.

[0077] Table 3 Note: Different letter subscripts after the data in the same column indicate significant differences (P<0.05).

[0078] Depend on Figure 2 As shown in Table 3, compared with the model group induced by dexamethasone alone, Examples 1-3 and Comparative Examples 1-6 significantly increased the relative expression levels of osteogenic markers ALP, RUNX2, Colla1, and OCN genes in osteoblasts, increased the relative expression level of the OPG gene, and significantly decreased the relative expression level of the RANKL gene. Compared with the model group, the relative expression level of the ALP gene in Comparative Example 7 was not significantly different, but it increased the relative expression levels of OPG, RUNX2, Colla1, and OCN genes and significantly decreased the relative expression level of the RANKL gene. Among them, the relative expression levels of osteogenic markers ALP, RUNX2, Colla1, OCN, and OPG genes in Example 2 were significantly higher than those in other examples, Comparative Examples 1-7, and the model group. The results from Example 2 and Comparative Examples 1-3 show that, under the same input of raw materials, compared with the processes of continuous ultrasound after enzymatic hydrolysis, continuous ultrasound followed by enzymatic hydrolysis, or no ultrasound and no enzymatic hydrolysis, the nutritional composition for improving bone density prepared by intermittent ultrasound treatment after enzymatic hydrolysis can significantly increase the relative expression of ALP and OPG genes in osteoblasts and decrease the relative expression of RANKL gene in osteoblasts. It can inhibit bone resorption by regulating the RANKL / OPG balance, further indicating that the nutritional composition has a promoting effect on increasing bone density and alleviating osteoporosis.

[0079] The statistical results of the relative gene expression levels of inflammatory factors IL-6, IL-1β, IL-12, and INOS in different groups are shown in Table 4. The statistical graph of the relative gene expression levels of inflammatory factors IL-6, IL-1β, IL-12, and INOS in different groups is shown below. Figure 3 As shown.

[0080] Table 4 Note: Different letter subscripts after the data in the same column indicate significant differences (P<0.05).

[0081] Depend on Figure 3As shown in Table 4, compared with the model group induced by dexamethasone alone, the relative expression levels of pro-inflammatory cytokines IL-6, IL-1β, IL-12, and INOS genes in osteoblasts were significantly reduced in groups 1-3 and 1-7. The nutritional composition for improving bone density showed better anti-inflammatory effects than the single ingredient. The results of group 2 and groups 1-3 show that, with the same input of each ingredient, compared with the processes of continuous sonication after enzymatic hydrolysis, continuous sonication followed by enzymatic hydrolysis, or no sonication and no enzymatic hydrolysis, the nutritional composition II for improving bone density, prepared by intermittent sonication after enzymatic hydrolysis, significantly inhibited the abnormally high expression of IL-6, IL-1β, IL-12, and INOS genes in osteoblasts, effectively reducing the level of cellular inflammatory factors and exhibiting excellent anti-inflammatory regulatory effects.

[0082] In summary, this invention provides a nutritional composition for improving bone density by combining kudzu root, colostrum basic protein, and selenium source. Verification has shown that this nutritional composition not only upregulates the expression of key osteogenic marker genes, effectively promoting osteoblast differentiation and accelerating bone formation, but also significantly inhibits the expression of pro-inflammatory factors and RANKL. By balancing the RANKL / OPG ratio to regulate bone metabolism, it effectively inhibits abnormal bone resorption and reduces local inflammatory responses in the bones. Therefore, the nutritional composition for improving bone density prepared in this invention can be used to prepare products for improving bone density and / or preventing osteoporosis, especially for preventing osteoporosis during pregnancy and lactation.

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nutritional composition for improving bone density, characterized in that, It includes kudzu root, colostrum basic protein, and selenium source.

2. The nutritional composition for improving bone density as described in claim 1, characterized in that, The mass ratio of kudzu root, colostrum basic protein, and selenium source is (200-1600):(50-200):(1-5); wherein the mass of selenium source is expressed as Se.

3. A method for preparing the nutritional composition for improving bone density according to claim 1 or 2, characterized in that, The preparation method includes the following steps: S1. Mix kudzu root with water and gelatinize to obtain a gelatinized dispersion; S2. Add cellulase to the gelatinized dispersion, perform enzymatic hydrolysis, and then subject it to intermittent ultrasonic treatment to obtain an ultrasonically treated solution; S3. Add colostrum basic protein and selenium source to the ultrasonic treatment solution to obtain the nutritional composition for improving bone density.

4. The method for preparing the nutritional composition for improving bone density as described in claim 3, characterized in that, The mass ratio of kudzu root to water is 1:8-12; and / or The gelatinization temperature is 85℃-95℃, and the gelatinization time is 20min-30min.

5. The method for preparing the nutritional composition for improving bone density as described in claim 3, characterized in that, Step S2 specifically includes the following steps: filtration of the gelatinized dispersion to obtain a filtrate; adjustment of the pH of the filtrate to 4.5-5.5 and the temperature to 45℃-55℃; addition of cellulase; incubation and enzymatic hydrolysis to obtain an enzymatic hydrolysate; inactivation of the enzyme in the enzymatic hydrolysate; cooling to 45℃-50℃; and intermittent ultrasonic treatment to obtain an ultrasonically treated solution; and / or The intermittent ultrasonic treatment has a power of 200W-300W and a frequency of 20kHz-40kHz; and / or The intermittent ultrasound treatment mode is as follows: each ultrasound treatment lasts 4-6 minutes, followed by a 1.5-2.5 minute pause; and / or The total processing time for the intermittent ultrasound is 25-45 minutes.

6. The method for preparing the nutritional composition for improving bone density as described in claim 3, characterized in that, Step S3 specifically includes the following steps: adding colostrum basic protein to the ultrasonic treatment solution, mixing evenly, adding selenium source, and then adding ascorbic acid to obtain the nutritional composition for improving bone density.

7. The use of the nutritional composition for improving bone density as described in claim 1 or 2, or the nutritional composition for improving bone density prepared by any one of claims 3-6, in the preparation of products for improving bone density.

8. The use of the nutritional composition for improving bone density as described in claim 1 or 2, or the nutritional composition for improving bone density prepared by any one of claims 3-6, in the preparation of products for the prevention or treatment of osteoporosis.

9. A product for improving bone density, characterized in that: The product contains the nutritional composition for improving bone density as described in claim 1 or 2.

10. A product for preventing and treating osteoporosis, characterized in that: The product contains the nutritional composition for improving bone density as described in claim 1 or 2.

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