A method for preparing hemp protein anti-osteoporosis peptide

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing hemp seed protein anti-osteoporosis peptides. The preparation method includes the following steps: drying hemp seeds at 40-50℃, pulverizing them, and passing them through a 40-80 mesh sieve; defatting them using supercritical fluid extraction to obtain defatted hemp seed material; mixing the defatted hemp seed material with water to form a homogenate, adjusting the pH to 9.5-10.5 with alkali, and extracting by stirring at 50-70℃ to obtain an alkaline extraction mixture. This invention uses alkaline and neutral proteases to enzymatically hydrolyze hemp seed protein stepwise, resulting in more complete hydrolysis compared to single-enzyme hydrolysis, and producing peptides with superior osteogenic activity. Cell experiments have demonstrated that this hemp seed protein peptide can increase the activity of alkaline phosphatase in osteoblasts, promote the formation of mineralized nodules, upregulate the expression of osteogenic-related genes, and accelerate osteogenic differentiation. Mechanistically, it activates the Smad / TGF-β / BMP pathway to regulate the expression of related proteins and promote osteogenic differentiation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing hemp protein anti-osteoporosis peptides. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass and deterioration of bone microstructure, significantly increasing bone fragility and fracture risk. It is a major public health issue in the context of global aging. With the continuous expansion of my country's elderly population, the prevalence of osteoporosis is rising, leading to increased medical expenditures and a growing social burden. While current clinical interventions for osteoporosis have clear efficacy, long-term use can cause various adverse reactions. Therefore, developing safe, efficient, and low-cost dietary intervention programs has become a research hotspot in the field of bone health. Hemp seed, one of the first plants recognized in my country as both food and medicine, is rich in high-quality plant protein and various bioactive components, possessing natural advantages for developing functional bone health products.

[0003] Existing extraction and enzymatic hydrolysis processes for hemp seed protein still have many shortcomings: conventional extraction processes have limited protein recovery rates and unstable retention rates of active ingredients; single enzymatic hydrolysis methods are insufficient to fully release small molecule active peptides, resulting in weak osteogenic activity of the products; at the same time, existing technologies have not yet formed standardized stepwise enzymatic hydrolysis processes for the targeted preparation of hemp protein peptides with osteoporosis-improving activity, nor have their mechanisms of action been systematically clarified, thus limiting the industrial application of hemp seeds in the field of bone health.

[0004] Therefore, the specific mechanisms of action of hemp seed protein and its hydrolysate in promoting osteogenic differentiation and treating osteoporosis have not been fully revealed, and there is a lack of technical solutions for preparing them into functional products specifically for the prevention and treatment of osteoporosis. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing hemp seed protein anti-osteoporosis peptides. The method employs stepwise enzymatic hydrolysis of hemp seed protein using alkaline and neutral proteases, resulting in more complete hydrolysis compared to single-enzyme hydrolysis. The peptides exhibit superior osteogenic activity. Cellular experiments have demonstrated that these hemp seed protein peptides can increase alkaline phosphatase activity in osteoblasts, promote the formation of mineralized nodules, upregulate the expression of osteogenic-related genes, and accelerate osteogenic differentiation. Mechanistically, they activate the Smad / TGF-β / BMP pathway to regulate the expression of related proteins and promote osteogenic differentiation.

[0006] The specific technical solution adopted by this invention is as follows: A method for preparing a hemp protein anti-osteoporosis peptide includes the following steps: Step 1: Degreasing treatment: Dry the hemp seeds at 40-50℃, crush them and pass them through a 40-80 mesh sieve. Then, degrease them by supercritical fluid extraction to obtain degreased hemp material. Step 2: Alkali extraction: Mix defatted hemp material with water to form a homogenate, adjust the pH to 9.5-10.5 with alkali, and extract by stirring at 50-70℃ to obtain an alkali extraction mixture; Step 3: Acid precipitation: The alkaline extraction mixture is subjected to solid-liquid separation. The liquid phase is taken and the pH is adjusted to 4.0-5.0 with acid to precipitate the protein. The supernatant is discarded, and the precipitated protein is collected and washed repeatedly with distilled water until a neutral pH is reached. Then, it is freeze-dried to obtain hemp seed protein. Step 4: First enzymatic hydrolysis: Dissolve the hemp seed protein in water again, add alkaline protease for first enzymatic hydrolysis. After the first enzymatic hydrolysis, heat at 90-100℃ for 3-10 minutes to inactivate the enzyme, and then cool to 40-50℃. Step 5: Secondary enzymatic hydrolysis: Adjust the pH to 6.5-7.5 using 1.0 mol / L HCl, add neutral protease for secondary enzymatic hydrolysis, and heat at 90-100℃ for 3-10 minutes after hydrolysis to inactivate the enzyme and obtain the enzymatic hydrolysate. Step 6: Separation and purification: Centrifuge the enzymatic hydrolysate, take the supernatant and filter it through a 0.22μm microporous membrane, collect the filtrate, and obtain hemp protein anti-osteoporosis peptide.

[0007] In a preferred embodiment, in step 1, the pressure of the supercritical fluid extraction is 20-40 MPa and the temperature is 35-50 °C.

[0008] In a preferred embodiment, in step 2, the ratio of the defatted hemp seed material to water is 1:8 to 1:10, the alkali is 1.0 mol / L food-grade NaOH, and the extraction time is 0.5 to 2 hours.

[0009] In a preferred embodiment, in step Step 3, the acid is 1.0 mol / L HCl.

[0010] In a preferred embodiment, in step 4, the enzymatic hydrolysis conditions for the first enzymatic hydrolysis are: temperature 45-55℃, pH 9.5-10.5, enzyme addition amount of 5000-10000 U / g substrate, and hydrolysis time of 3-5 hours.

[0011] In a preferred embodiment, in step 5, the enzymatic hydrolysis conditions for the secondary enzymatic hydrolysis are: temperature 40-50℃, enzyme addition amount of 5000-10000 U / g substrate, and hydrolysis time of 3-5 hours.

[0012] In a preferred embodiment, in step 6, the centrifugation temperature is 0–10°C, the rotation speed is 8000–12000 rpm, and the centrifugation time is 15–25 min.

[0013] A hemp protein anti-osteoporosis peptide, prepared by any of the above-described preparation methods.

[0014] The hemp protein anti-osteoporosis peptide promotes osteoblast differentiation and bone mineralization by activating the Smad / TGF-β / BMP signaling pathway, upregulating the expression of osteogenic-related genes and proteins.

[0015] The hemp protein anti-osteoporosis peptide is used to increase alkaline phosphatase (ALP) activity and mineralized nodule formation, and upregulate the expression levels of osteogenic-related genes Alp and Runx2.

[0016] The hemp protein anti-osteoporosis peptide is used to increase bone mineral density (BMD), bone volume to total volume ratio (BV / TV), number of trabeculae (Tb.N) and bone mineral content (BMC), reduce trabecular separation (Tb.Sp), and increase mineral deposition rate (MAR).

[0017] The application of the hemp protein anti-osteoporosis peptide in the preparation of drugs or functional foods that promote osteogenic differentiation and prevent osteoporosis.

[0018] The technical effects achieved by this invention are as follows: This invention uses an alkaline extraction and acid precipitation method combined with stepwise enzymatic hydrolysis to extract and enzymatically hydrolyze hemp seeds to obtain hemp protein peptides with osteogenic differentiation-promoting activity. The preparation method is stable, simple to operate, and suitable for industrial production. This invention employs a stepwise enzymatic hydrolysis strategy using alkaline and neutral proteases, which, compared to a single enzymatic hydrolysis method, can more fully hydrolyze hemp seed protein, yielding a peptide composition with stronger biological activity and effectively improving the product's osteogenic activity. This invention demonstrates through cell experiments that the hemp protein anti-osteoporosis peptide can significantly enhance alkaline phosphatase activity and mineralized nodule formation in MC3T3-E1 cells and human mesenchymal stem cells (hMSCs), upregulate the expression levels of osteogenic-related genes Alp and Runx2, thereby effectively promoting osteoblast differentiation. Through mechanistic studies, this invention has discovered that the hemp protein anti-osteoporosis peptide activates the Smad / TGF-β / BMP signaling pathway, upregulates the expression levels of Smad4, BMP2 and BMP4, while inhibiting the expression of Smad2 / 3, thus promoting osteogenic differentiation and providing a new molecular mechanism for targeted treatment of osteoporosis. This invention demonstrates, through ovariectomy (OVX) mouse models and aged mouse models, that the hemp protein anti-osteoporosis peptide can significantly increase mineral deposition rate, bone mineral density and bone mineral content, increase the number of trabeculae, reduce trabecular separation, effectively counteract ovariectomy and aging-induced bone loss, and delay the progression of osteoporosis. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the relative cell viability of hMSCs in Test Example 1 of this invention; Figure 2 This is a schematic diagram of the ALP and ARS staining results of hMSCs in Test Example 2 of this invention; Figure 3 This is a schematic diagram of the ALP and ARS staining results of MC3T3-E1 cells in Test Example 2 of this invention; Figure 4 This is a schematic diagram of the relative expression levels of osteogenic genes Alp and Runx2 mRNA in hMSCs detected by RT-PCR in Test Example 3 of this invention; Figure 5 This is a schematic diagram showing the relative expression levels of osteogenic genes Alp and Runx2 mRNA in MC3T3-E1 cells in test example three of this invention; Figure 6 This is a schematic diagram illustrating the detection of osteogenic-related transcription factor mRNA expression levels in MC3T3-E1 cells by RT-PCR in Test Example 4 of this invention; Figure 7 This is a schematic diagram of the mRNA expression levels of key transcription factors in hMSCs in test example four of this invention; Figure 8 This is a graph showing the results of dual-luciferase reporter gene detection of transcription factor activity in MC3T3-E1 cells in Test Example 4 of this invention; Figure 9 This is a graph showing the results of dual-luciferase reporter gene detection of hMSCs transcription factor activity in Test Example 4 of this invention; Figure 10 This is a graph showing the effect of HSP / HPH on osteogenic differentiation of hMSCs and expression of Alp / Runx2 genes under the intervention of the Wnt pathway inhibitor NC043 in Test Example 5 of this invention; Figure 11 This is a graph showing the effect of HSP / HPH on osteogenic differentiation of hMSCs and expression of Alp / Runx2 genes under the intervention of BMP pathway inhibitor DM-3189 in Test Example 5 of this invention; Figure 12 This is a figure showing the results of Western blot and RT-PCR detection of the effects of HSP / HPH on the expression of SMAD4 / 5 and BMP2 / 4 proteins and genes in MC3T3-E1 and hMSCs in Test Example 5 of this invention; Figure 13 This is a schematic diagram of the RT-PCR detection of the expression levels of Smad2 and Smad3 mRNA in MC3T3-E1 and hMSCs by HSP / HPH in test example five of this invention; Figure 14This is a diagram showing the results of mineral deposition, bone microstructure, and bone marrow osteogenic gene detection in OVX osteoporotic mice in Test Example 6 of this invention; Figure 15 These are pathological sections of liver, spleen, and kidney H&E from OVX mice and biochemical indicators of serum ALT / AST / BUN in Test Example 6 of this invention. Figure 16 This is a graph showing the results of mineral deposition, bone microstructure, and bone marrow osteogenic gene detection in aged osteoporotic mice in Test Example 7 of this invention; Figure 17 This is a graph showing the H&E pathological sections of the liver, spleen, and kidney of aged mice and the detection of serum ALT / AST / BUN biochemical indicators in Test Example 7 of this invention. Figure 18 This is a flowchart illustrating the preparation process of hemp protein anti-osteoporosis peptide in this invention. Detailed Implementation

[0020] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0022] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in a preferred embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments.

[0023] Example:

[0024] Please see Figure 18 As shown in the figure, this embodiment provides a method for preparing hemp protein anti-osteoporosis peptide, and the specific steps are as follows: Hemp seeds were dried in a 45℃ oven to constant weight, then pulverized and passed through a 60-mesh sieve. Supercritical fluid extraction (30MPa, 45℃) was used to remove oil, yielding defatted hemp seed material. This defatted hemp seed material was mixed with distilled water at a material-to-liquid ratio of 1:9 and homogenized using a colloid mill. The pH was adjusted to 10.0 with 1.0 mol / L food-grade NaOH, and the mixture was continuously stirred in a 60℃ water bath for 1 hour to obtain an alkaline extraction mixture. This mixture was centrifuged at 10,000 rpm for 15 minutes for solid-liquid separation. The liquid phase was adjusted to pH 4.5 with 1.0 mol / L HCl and allowed to stand for 30 minutes to allow protein precipitation. The protein precipitate was collected by centrifugation (discarding the supernatant). The collected protein precipitate was repeatedly washed with distilled water until the pH was neutral, and then freeze-dried to obtain... Hemp seed protein was obtained by redissolving the freeze-dried hemp seed protein in water. Alkaline protease was added at a ratio of 8000 U / g substrate, and the pH of the system was adjusted to 10.0. The system was then subjected to a first enzymatic hydrolysis at 50°C for 4 hours. After the first enzymatic hydrolysis, the temperature was raised to 95°C and heated for 5 minutes to inactivate the enzyme. After cooling to 45°C, the pH of the system was adjusted to 7.0, and neutral protease was added at a ratio of 8000 U / g substrate. A second enzymatic hydrolysis was performed at 45°C for 4 hours. After the second enzymatic hydrolysis, the temperature was raised to 95°C again and heated for 5 minutes to inactivate the enzyme, yielding the enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged at 10000 rpm for 20 minutes at 4°C. The supernatant was filtered through a 0.22 μm microporous membrane for sterilization to obtain hemp protein anti-osteoporosis peptide. The hemp protein anti-osteoporosis peptide was then stored at -20°C.

[0025] In this embodiment, hemp seed protein is extracted by alkaline extraction and acid precipitation, and then the large molecular weight hemp seed protein is hydrolyzed into small molecular active peptides by a stepwise double enzymatic hydrolysis process using alkaline protease and neutral protease. The hydrolysis products can upregulate the expression of osteogenic-related genes and proteins by activating the BMP / Smad signaling pathway, thereby promoting osteoblast differentiation and mineralization and exerting a bone formation promoting effect. Compared with the single enzymatic hydrolysis method, hemp seed protein can be hydrolyzed more completely to obtain a peptide composition with stronger biological activity.

[0026] Test example: Test Example 1: Effect of Hemp Protein Anti-Osteoporosis Peptide on Cell Viability Human mesenchymal stem cells (hMSCs, purchased from Procell, CP-H166) in the logarithmic growth phase were harvested at a density of 6 × 10⁶ cells / well. 3Cells were seeded at a density of 100 μg / mL in 96-well plates and incubated at 37°C for 24 hours in a 5% CO2 incubator. Hemp seed protein anti-osteoporosis peptide (HPH) and hemp seed protein (HSP) prepared in the previous example were added to the culture medium to a final concentration of 50 μg / mL. Cells treated with an equal volume of PBS served as a control group. After 48 hours of treatment, the cells were washed three times with phosphate-buffered saline (PBS), and 100 μL of culture medium containing 10 μL of CCK-8 dye (Cell Counting Kit-8, BS350B, Beijing Lanjike Technology Co., Ltd.) was added to each well. Incubation was continued for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Each group had 6 independent replicates. The test results are shown in Table 1 and 2. Figure 1 As shown.

[0027] Table 1: ; Note: Refer to Table 1 and Figure 1 As can be seen, there was no significant difference in cell viability between the HSP group and the HPH group and the control group (p>0.05), indicating that the hemp protein anti-osteoporosis peptide prepared in the examples has no obvious cytotoxicity to hMSCs and has good biosafety.

[0028] Test Example 2: Test on the promoting effect of hemp protein anti-osteoporosis peptide on osteogenic differentiation (ALP staining and ARS staining) Pre-osteoblasts and hMSCs from MC3T3-E1 mice were collected at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well plates and cultured in osteogenic differentiation medium (composed of DMEM, 10% FBS, 1% penicillin / streptomycin, 5 mmol / L). -1 β-glycerophosphate sodium, 50 μg·mL -1 Cells were cultured in ascorbic acid and 1% L-glutamine to induce osteogenic differentiation. Cells were treated with 50 μg / mL HSP or HPH, with an equal volume of PBS used as a control. ALP staining and Alizarin Red S (ARS) staining were performed after 3 and 21 days of osteogenic induction culture. ALP staining was performed using the BCIP / NBT staining kit: cells were washed three times with PBS, fixed with 10% formaldehyde, washed with PBS, and incubated with the BCIP / NBT mixture for 30 minutes. When the cells turned blue or purple, staining was terminated by washing with deionized water. After drying at room temperature, the staining was recorded using a CanoScan 9000F Mark II scanner. ARS staining involved washing cells with PBS, incubating with 0.5% Alizarin Red S (pH 4.2, A5533, Sigma-Aldrich) for 30 minutes, soaking in water for 30 minutes, drying, and scanning. Results are shown in [reference needed]. Figures 2 to 3 As shown.

[0029] Combination Figure 2 and Figure 3 The staining results show that the ALP activity of MC3T3-E1 cells and hMSCs in both the HSP and HPH treatment groups was significantly enhanced, and the formation of mineralized nodules was significantly increased. The HPH group showed a more significant effect, indicating that the hemp protein anti-osteoporosis peptide prepared in this invention can effectively promote osteoblast differentiation and mineralization.

[0030] Test Example 3: Effect of Hemp Seed Protein Anti-Osteoporosis Peptide on Osteogenesis-Related Gene Expression MC3T3-E1 cells and hMSCs were collected at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well plates and treated with 50 μg / mL HSP or HPH for 24 hours (for RNA extraction). Cells treated with an equal volume of PBS served as a control. Total RNA was extracted using the EZNA® Total RNA Kit I (R6834-02, Omega Bio-TEK) and reverse transcribed into cDNA using the HiScript® II 1st Strand cDNA Synthesis Kit (R211-02, Vazyme). RT-PCR was performed using ChamQ Universal SYBR Quantitative PCR Premix (Q711-02-AA, Vazyme) and a Roche 480 II Real-Time PCR System, with Gapdh as an internal control. The relative mRNA expression level was calculated using the 2⁻ΔΔCt method. Primer sequences for osteogenic marker genes Alp and Runx2 are shown in Table 2. The test results are shown in Table 3. Figure 4 as well as Figure 5 As shown.

[0031] Table 2: ; Table 3: ; Note: Compared with the control group, p < 0.01. .

[0032] Combining Table 2 and Table 3, Figure 4 as well as Figure 5 As can be seen, compared with the control group, the mRNA expression levels of Alp and Runx2 in MC3T3-E1 cells and hMSCs were significantly increased after HSP and HPH treatment (p < 0.01 or p < 0.001), with the HPH group showing a more significant upregulation, indicating that the hemp protein anti-osteoporosis peptide prepared in the examples can activate the osteogenic program at the transcriptional level.

[0033] Test Example 4: Effects of Hemp Seed Protein Anti-Osteoporosis Peptide on the Regulation of Osteogenesis-Related Transcription Factors MC3T3-E1 cells and hMSCs were collected at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 mcg / mL in 6-well plates. After treatment with 50 μg / mL HSP or HPH for 24 hours, cells were harvested and RNA was extracted. The mRNA expression levels of key transcription factors such as TCF7, SMAD4, FOXO1, HES1, CEBP, HIF1, NFKB, and SMAD2 were detected by RT-PCR. Simultaneously, 1 × 10⁻⁶ cells per well were seeded. 5 Cells were cultured at a density of 1000 cells per well in 6-well plates. 0.4 μg of Topflash luciferase plasmid and 0.4 μg of Renilla luciferase plasmid were transfected into cells in Opti-MEM containing 50 μg / mL HSP / HPH. After 72 hours of culture, the cells were detected using a dual luciferase assay system. The luminescence signal was quantified using a microplate reader and normalized to Renilla luciferase activity.

[0034] RT-PCR results showed that HSP / HPH treatment significantly upregulated the expression of Tcf7 and Smad4 in MC3T3-E1 cells, while reducing the expression of Hes1, Cebp, Nfkb, and Smad2 (e.g., Figure 6 As shown); consistent results were obtained in hMSCs (e.g. Figure 7 As shown). The results of luciferase reporter gene detection were consistent with the RT-PCR results (e.g., Figure 8 and Figure 9 (As shown). The above results indicate that the hemp protein anti-osteoporosis peptide prepared in this invention regulates the osteogenic microenvironment through a dual mechanism: on the one hand, it activates the osteogenic program through TCF7 and SMAD4, and on the other hand, it reduces differentiation resistance by inhibiting HES1, CEBP, NFKB and SMAD2.

[0035] It should be noted that, Figure 6 and Figure 7 The key transcription factors corresponding to A through H are, in order: Tcf7, Smad4, Pparg, Hes1, Cebp, Hif1, Nfκb, and Smad2. Figure 8 and Figure 9 The key transcription factors corresponding to I to P are, in order: Tcf7, Smad4, Pparg, Hes1, Cebp, Hif1, Nfκb, and Smad2.

[0036] Test Example 5: Verification Test on the Mechanism of Hemp Seed Protein Anti-Osteoporosis Peptide Regulating Osteogenic Differentiation Through the TGF-β / BMP Signaling Pathway Take hMSCs at a ratio of 3 × 10⁻⁶ per well 5Cells were seeded at a density of [number] cells per well in 6-well plates and divided into three groups: a DMSO control group, a BMP signaling inhibitor DM-3189 treatment group, and a Wnt / β-catenin pathway inhibitor NC043 treatment group. Cells in each group were supplemented with 50 μg / mL HSP or HPH along with the corresponding inhibitor. After osteogenic induction culture for 3 and 21 days, ALP and ARS staining were performed, and RNA was extracted for RT-PCR detection of Alp and Runx2 mRNA expression. The results are as follows: Figure 10 and Figure 11 As shown; combined with Figure 10 and Figure 11 It can be seen that in the presence of NC043 (a Wnt inhibitor), HSP / HPH treatment still significantly promoted ALP activity and mineralized nodule formation, and the expression of both Alp and Runx2 was significantly increased (e.g., Figure 10 ); however, under DM-3189 (BMP signaling inhibitor) treatment, the osteogenic effect of HSP / HPH was significantly weakened (e.g. Figure 11 ); Simultaneously, MC3T3-E1 cells and hMSCs were collected at a ratio of 3 × 10⁶ cells per well. 5 Cells were seeded at a density of 1,000 cells per well in 6-well plates. After treatment with 50 μg / mL HSP or HPH for 48 hours, cells were collected and proteins were extracted. Cells were digested with cell lysis buffer (P0013, Beyotime), and a 1% protease inhibitor mixture (539134, Calbiochem) was added. After lysis on ice for 30 minutes, the cells were centrifuged at 13000g for 15 minutes to extract proteins. Quantification was performed using the BCA method (TaKaRa). An equal volume of protein was loaded onto a sample for SDS-PAGE electrophoresis (electrophoresis conditions: 80 volts for 30 minutes, followed by 120 volts for 90 minutes). The protein was transferred to a nitrocellulose membrane (66485, Pall, 250mA, 2.5h), blocked with 5% skim milk for 60 min, and incubated with the primary antibody at 4°C for 15 hours. After incubation with the HRP-labeled secondary antibody (1:5000, HP0511, HUABIO, China), the cells were developed using a chemiluminescence detection system, and the grayscale values ​​were analyzed using ImageJ. Western blot results showed that HSP / HPH treatment significantly upregulated the protein expression of SMAD4, SMAD5, BMP2, and BMP4 (e.g., Figure 12 -A and Figure 12 -D), RT-PCR detection further confirmed that the mRNA levels of Bmp2 and Bmp4 were significantly elevated (e.g., Figure 12 -B、 Figure 12 -C、 Figure 12 -E、 Figure 12 -F indicates that the expression of Smad2 and Smad3 was significantly reduced (as shown in the figure). Figure 13As shown above, the results indicate that the hemp protein anti-osteoporosis peptide prepared in the examples mainly promotes osteogenic differentiation by activating the TGF-β / BMP signaling pathway (upregulating the expression of Smad4, BMP2 and BMP4, while inhibiting the expression of Smad2 / 3).

[0037] in, Figure 10 Figure A shows a comparison of ALP and ARS staining results in human mesenchymal stem cells (hMSCs) treated with HSP / HPH (with the addition of DMSO or NCO43 inhibitors). Figure 10 Figure B is a schematic diagram comparing the expression levels of Alp in human mesenchymal stem cells treated with HSP / HPH (with the addition of DMSO or NCO43 inhibitors); Figure 10 The diagram in C is a comparison of Runx2 mRNA expression levels in human mesenchymal stem cells treated with HSP / HPH (with the addition of DMSO or NCO43 inhibitors). Figure 11 The image in middle H shows a comparison of ALP and ARS staining results in human mesenchymal stem cells (hMSCs) treated with HSP / HPH (with the addition of DMSO or DM-3189 inhibitors). Figure 11 Figure I is a schematic diagram comparing the expression levels of Alp in human mesenchymal stem cells treated with HSP / HPH (with the addition of DMSO or DM-3189 inhibitors); Figure 11 The image in J is a schematic diagram comparing the expression levels of Runx2 mRNA in human mesenchymal stem cells treated with HSP / HPH (with the addition of DMSO or DM-3189 inhibitors). Figure 12 Figure A shows a schematic diagram of the expression levels of SMAD4, SAMD5, BMP2 and BMP4 in MC3T3-E1 cells detected by Western blotting. Figure 12 Figure B is a schematic diagram illustrating the detection of Bmp2 expression levels in MC3T3-E1 cells by RT-PCR. Figure 12 The diagram in C is a schematic diagram of the expression level of Bmp4 in MC3T3-E1 cells detected by RT-PCR. Figure 12 The diagram in middle D is a schematic diagram of the expression levels of SMAD4, SAMD5, BMP2 and BMP4 in human mesenchymal stem cells (hMSCs) detected by Western blotting. Figure 12 E in the diagram represents the mRNA expression level of Bmp2 in hMSCs; Figure 12The diagram in F shows the mRNA expression level of Bmp4 in hMSCs; Figure 13 A in the diagram represents the mRNA level of Smad2 in MC3-3-E1 cells; Figure 13 Figure B shows the mRNA level of Smad3 in MC3-3-E1 cells; Figure 13 The diagram in C represents the mRNA level of Smad2 in hMSCs; Figure 13 The diagram in D shows the mRNA level of Smad3 in hMSCs.

[0038] It should be noted that the first antibody used in this test case is: GAPDH Rabbit pAb(1:5000,ET1601-4,HUABIO, China). ALP Rabbit pAb(1:2000,ET1601-21,HUABIO, China). RUNX2 Rabbit pAb(1:2000,ET1612-47,HUABIO, China). BMP2 Rabbit pAb(1:1000,CSB-PA09419A0Rb,CUSABIO,China). BMP4 Rabbit pAb(1:1000,CSB-PA05799A0Rb,CUSABIO,China). SMAD4 Rabbit pAb (1:1000, CPA5550, Cohesion, China) SMAD5 Rabbit pAb (1:1000, CPA1710, Cohesion, China); Test Example 6: Therapeutic Effect of Hemp Protein Anti-Osteoporosis Peptide on Ovarian Removal (OVX) Osteoporosis Mice Fifty-four 3-month-old female C57BL / 6 mice (23.7±0.5g) underwent ovariectomy (OVX) or sham surgery and were randomly divided into 6 groups (n=9 per group): baseline group (BL), sham surgery group, OVX group, OVX+PBS group, OVX+HSP group, and OVX+HPH group. Mice were anesthetized with 1.5% sodium pentobarbital (1 mL per 100g body weight). A 0.8cm incision was made at the costal angle along the midline, the peritoneum was bluntly dissected, and the ovary was located and removed along the uterine branch (in the sham surgery group, only a segment of fat was removed). Postoperatively, the area was disinfected with erythromycin ointment. OVX treatment began 15 days post-operation via gavage. The HSP and HPH groups received 10mg / kg of HSP or HPH solution daily, divided into two doses, for one week. The PBS group received 400μL twice daily. PBS; the remaining groups received no treatment; mice were sacrificed 35 days after OVX, and skull and femur samples were collected. Bone marrow cells (BMCs) were extracted for RT-PCR detection. Calcein (20 mg / kg body weight) was injected intraperitoneally 12 and 3 days before sacrifice for double labeling. Skull samples were fixed in 4% paraformaldehyde and embedded in OCT. 4 μm cross-sectional frozen sections were prepared using a cryostat and observed under a fluorescence microscope. Dynamic bone histomorphometry analysis (MAR) was performed using ImageJ software. Femur samples were scanned using a micro-CT system. Isotropic voxels with a size of 10.5 μm (70 kVp, 114 mA, 200 ms integration time) were used for reconstruction analysis using SCANCO software. Bone mineral density (BMD), bone volume / tissue volume ratio (BV / TV), trabecular bone number (Tb.N), trabecular bone separation (Tb.Sp), and bone mineral content (BMC) were calculated using CTvol software. Simultaneously, mouse liver, spleen, and kidneys were stained with hematoxylin and eosin (H&E). Serum samples were collected from the orbital fossa to detect ALT, AST, and BUN levels to assess biosafety. The test results are shown in Table 4. Figure 14 As shown.

[0039] Table 4: ; Note: Compared with the sham surgery group, ###p<0.001; compared with the OVX+PBS group, , .

[0040] Compared with the sham-operated group, OVX mice showed significantly decreased BMD, BV / TV, Tb.N, and BMC, and significantly increased Tb.Sp (p < 0.001). Compared with the OVX+PBS group, HSP and HPH interventions significantly increased BMD, BV / TV, Tb.N, and BMC, and significantly decreased Tb.Sp (p < 0.05 or p < 0.01), with the HPH group showing more significant effects (e.g., ...). Figure 14 C to Figure 14 H); Calcein double labeling showed a significant increase in MAR in both the HSP and HPH groups (e.g., Figure 14 A and Figure 14 B); The mRNA expression levels of Alp and Runx2 in BMCs were significantly increased in both the HSP and HPH groups (e.g., Figure 14 I and Figure 14 J); H&E staining showed no obvious morphological abnormalities in the liver, spleen, and kidneys, and serum ALT, AST, and BUN levels were all within safe ranges (e.g., Figure 15 A to Figure 15 (D) The above results indicate that the hemp protein anti-osteoporosis peptide prepared in the examples can effectively and safely salvage OVX-induced bone loss.

[0041] Test Example 7: Therapeutic Effect Test of Hemp Protein Anti-Osteoporosis Peptide on Aged Osteoporosis Mice Eighteen 6-month-old male C57BL / 6 mice (25.2±0.5g) were raised to 18 months of age and then randomly divided into three groups (n=6 per group): PBS group, HSP group, and HPH group. Mice were administered PBS, HSP, or HPH by gavage at a dose of 10 mg / kg once daily for 20 days. Four weeks later, the mice were sacrificed, and bone marrow, skull, and femur samples were collected. Calcein double labeling, micro-CT scanning analysis, bone histomorphometric analysis, RT-PCR detection, and biosafety testing were performed using the same methods as in Test Case 6. The test results are shown in Table 5. Figure 16 and Figure 17 As shown, compared with the PBS group, the BMD, BV / TV, Tb.N, and BMC of aged mice in the HSP and HPH groups were significantly increased, while Tb.Sp was significantly decreased (p < 0.05 or p < 0.01), with the HPH group showing more significant improvement (e.g., ...). Figure 16 D to H); Calcein double labeling showed that HSP and HPH treatment significantly increased MAR in aged mice (e.g., Figure 16 A and Figure 16 B); Microscopic CT three-dimensional reconstruction images visually show that the trabecular structure of bone is significantly improved after HSP / HPH treatment (e.g. Figure 16 C); The mRNA expression levels of Alp and Runx2 in BMCs were significantly upregulated in both the HSP and HPH groups (e.g., Figure 16 I and Figure 16 J); Biochemical safety testing and histological examination of major organs revealed no abnormalities (e.g. Figure 17 A to Figure 17 (D) The above results indicate that the hemp protein anti-osteoporosis peptide prepared in the examples can effectively reverse osteoporosis in aged mice and has good in vivo safety.

[0042] Table 5: ; Note: Compared with the control group, , .

[0043] Test Example 8: In vivo biocompatibility test of hemp protein anti-osteoporosis peptide In Test Cases 6 and 7, blood was collected from the orbital region of mice before sacrifice. Serum was separated by centrifugation at 14000g for 12 minutes. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) were detected using an ADVIA 2400 chemical analysis system. Simultaneously, liver, spleen, and kidney tissues were collected from mice, fixed in 4% paraformaldehyde at 4°C for 48 hours, dehydrated, embedded in paraffin, and cut into 5μm sections. Hematoxylin staining for 5 minutes, eosin staining for 20 seconds, and neutral resin mounting were performed. Images were acquired using a high-throughput slide scanning imaging system. The test results are shown in Table 6. There were no significant differences in serum ALT, AST, and BUN levels between the drug-treated groups and the control group (p > 0.05), and all levels were within the normal physiological range. H&E staining showed no obvious morphological abnormalities in the liver, spleen, and kidneys (e.g., ...). Figure 15 A, Figure 17 A) The above results indicate that the hemp protein anti-osteoporosis peptide prepared in the examples has good biocompatibility under in vivo administration conditions.

[0044] Table 6: ; Based on the test results of Test Examples 1 to 8 above, it can be seen that: a. Safety: The hemp protein anti-osteoporosis peptide prepared in the examples had no obvious cytotoxicity to hMSCs (Test Example 1). After administration to OVX mice and aged mice, serum ALT, AST and BUN levels were all within the normal range, and no obvious morphological abnormalities were observed in liver, spleen and kidney H&E staining (Test Example 8), indicating that the peptide has good in vitro and in vivo biological safety. b. Regarding the activity of promoting osteodifferentiation: The hemp protein anti-osteoporosis peptide prepared in the examples can significantly enhance the activity of ALP and the formation of mineralized nodules in MC3T3-E1 cells and hMSCs (test example 2), and significantly upregulate the mRNA expression levels of osteogenic related genes Alp and Runx2 (test example 3), indicating that it has significant activity of promoting osteodifferentiation, and the activity of HPH is better than that of HSP. c. Mechanism of action: The hemp protein anti-osteoporosis peptide prepared in the examples regulates osteogenic differentiation through a dual mechanism—activating the osteogenic program through TCF7 and SMAD4, while inhibiting HES1, CEBP, NFKB, and SMAD2 to reduce differentiation resistance (Test Example 4); further mechanistic studies showed that the peptide mainly works by activating the TGF-β / BMP signaling pathway, specifically by upregulating the expression of SMAD4, SMAD5, BMP2, and BMP4, while inhibiting the expression of Smad2 / 3 (Test Example 5). d. In vivo efficacy: The hemp protein anti-osteoporosis peptide prepared in the examples significantly increased MAR, BMD, BV / TV, Tb.N and BMC and decreased Tb.Sp in both the OVX osteoporosis mouse model (test example six) and the aged osteoporosis mouse model (test example seven), effectively counteracting ovariectomy and aging-induced bone loss and delaying the progression of osteoporosis. Moreover, the therapeutic effect of HPH was better than that of HSP.

[0045] In summary, the hemp protein anti-osteoporosis peptides prepared by the alkaline extraction, acid precipitation, and stepwise enzymatic hydrolysis process in this application exhibit significant osteogenic differentiation-promoting activity and anti-osteoporosis effects, with good safety profile. They can be used to prepare drugs or functional foods that promote osteogenic differentiation and prevent osteoporosis. The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described or explained in this invention are implemented according to conventional methods in the art unless otherwise specified or limited.

Claims

1. A method for preparing hemp protein anti-osteoporosis peptide, characterized in that: Includes the following steps: Step 1: Dry the hemp seeds at 40-50℃, grind them and pass them through a 40-80 mesh sieve. Then, degrease them by supercritical fluid extraction to obtain defatted hemp seed material. Step 2: Mix the defatted hemp material with water to form a homogenate, adjust the pH to 9.5-10.5 with alkali, and extract by stirring at 50-70℃ to obtain an alkali-extracted mixture; Step 3: Perform solid-liquid separation on the alkaline extraction mixture, take the liquid phase and adjust the pH to 4.0-5.0 with acid to precipitate the protein, discard the supernatant, collect the precipitated protein portion and wash it repeatedly with distilled water until a neutral pH is reached, and then freeze-dry it to obtain hemp seed protein; Step 4: Redissolve the hemp seed protein in water, add alkaline protease for one enzymatic hydrolysis, heat at 90-100℃ for 3-10 minutes after the first enzymatic hydrolysis, and then cool to 40-50℃. Step 5: Adjust the pH to 6.5-7.5 with 1.0 mol / L HCl, add neutral protease for secondary enzymatic hydrolysis, and heat at 90-100℃ for 3-10 minutes after enzymatic hydrolysis to obtain the enzymatic hydrolysate; Step 6: Centrifuge the enzymatic hydrolysate, take the supernatant and filter it through a 0.22μm microporous membrane, collect the filtrate, and obtain hemp protein anti-osteoporosis peptide.

2. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In Step 1, the pressure of the supercritical fluid extraction is 20-40 MPa and the temperature is 35-50 °C.

3. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In Step 2, the ratio of defatted hemp seed material to water is 1:8 to 1:10, the alkali is food-grade NaOH, and the extraction time is 0.5 to 2 hours.

4. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In step 3, the acid is 1.0 mol / L HCl.

5. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In step 4, the enzymatic hydrolysis conditions for the first enzymatic hydrolysis are: temperature 45-55℃, pH 9.5-10.5, enzyme addition amount of 5000-10000 U / g substrate, and enzymatic hydrolysis time of 3-5 hours.

6. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In step 5, the enzymatic hydrolysis conditions for the secondary enzymatic hydrolysis are: temperature 40-50℃, enzyme addition amount of 5000-10000 U / g substrate, and hydrolysis time of 3-5 hours.

7. The method for preparing a hemp protein anti-osteoporosis peptide according to claim 1, characterized in that: In Step 6, the centrifugation temperature is 0–10℃, the rotation speed is 8000–12000 rpm, and the centrifugation time is 15–25 min.

8. A hemp protein anti-osteoporosis peptide, characterized in that, It is prepared by any one of claims 1 to 7.

9. The hemp protein anti-osteoporosis peptide according to claim 8, characterized in that: The hemp protein anti-osteoporosis peptide promotes osteoblast differentiation and bone mineralization by activating the Smad / TGF-β / BMP signaling pathway, upregulating the expression of osteogenic-related genes and proteins.

10. The application of the hemp protein anti-osteoporosis peptide as described in claim 8 in the preparation of drugs or functional foods that promote osteogenic differentiation and prevent osteoporosis.

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