Use of mulberry total polyphenol extract in preparation of product for promoting bone formation

Abstract

Provided is the use of a mulberry total polyphenol extract in the preparation of a product for promoting bone formation. The active ingredients of the mulberry total polyphenol extract include phenolic acids, polyphenol derivatives, flavones, isoflavones and tannins. The product for promoting bone formation can promote osteoblast proliferation, promote osteogenic calcification, inhibit sclerostin activity, and / or promote bone growth.

Description

Application of Mulberry Total Polyphenol Extract in the Preparation of Products Promoting Bone Formation Technical Field

[0001] This invention relates to the field of pharmaceutical and health food application technology, specifically to the application of a total polyphenol extract of mulberry in the preparation of products that promote bone formation. Background Technology

[0002] With the aging population, the incidence of bone-related diseases such as fractures and osteoporosis continues to rise, becoming a global concern and creating a huge market demand for bone formation-promoting products. From early estrogen receptor modulators, bisphosphonates, and human parathyroid hormone analogs (such as teriparatide) to the recent global benchmarks of bone formation-promoting monoclonal antibody drugs, such as denosumab and romosozumab, significant progress has been made in preventing and controlling fractures. However, existing drugs have many shortcomings that affect their clinical application. For example, mainstream drugs like teriparatide and romosozumab require injection, leading to poor patient compliance, extremely high costs, and numerous cardiovascular and cerebrovascular side effects. Therefore, finding more practical and safer innovative drugs to promote bone formation has always been a global research hotspot.

[0003] In 2019, the FDA approved Evenity, a new osteosclerosing protein inhibitor, for marketing in the United States, making it the first new anti-osteoporosis drug with a dual effect of increasing bone formation and reducing bone resorption. Osteosclerosing protein is a glycoprotein encoded by the Sost gene and secreted by osteogenic cells. Inhibiting its expression or activity can significantly increase bone mass and bone strength without significant extraosseous adverse reactions, thus making it an effective therapeutic target for osteoporosis. Animal models and clinical studies have shown that osteosclerosing protein inhibitors have significant advantages over traditional drugs such as alendronate sodium and teriparatide.

[0004] Traditional Chinese medicine, accumulated over thousands of years, is an inexhaustible treasure trove for new drug discoveries. Mulberry, a traditional Chinese medicine with a long history of use, is also listed in the National Health Commission's catalog of medicinal and edible herbs. Mulberry enters the kidney meridian; the kidney governs bones and produces marrow. Classic records of mulberry treating bone and joint-related diseases date back to ancient times, such as in the Tang Dynasty's *Compendium of Materia Medica* (*Bencao Shiyi*): "It stops thirst and benefits the five internal organs and joints"; in the Ming Dynasty's *Compendium of Materia Medica* (*Bencao Gangmu*): "It benefits the five internal organs and joints and promotes blood circulation"; and in the *Essentials of Materia Medica* (*Bencao Cuoyao*): "It is particularly effective in replenishing water and benefiting the five internal organs and joints." These classic medical texts provide valuable evidence-based medicine. However, to date, we still cannot fully and accurately understand the material basis and mechanism of the above-mentioned efficacy of mulberry from the perspective of modern scientific understanding.

[0005] Although modern TCM research has revealed that mulberry total polyphenols have multiple biological effects, including anti-oxidation, hypoglycemia, blood lipid regulation, and anti-atherosclerosis (Research progress on the functional properties of mulberry total polyphenol extract, Light Industry Science and Technology, Vol. 36, No. 10, 2020; Research progress on functional components of mulberry and their development and utilization, Food Safety Guide, 2021(33)); at the same time, research on the compound composition of mulberry total polyphenols is still ongoing (Two-dimensional liquid chromatography separation and identification of polyphenolic compounds in mulberry, Journal of Food Safety and Quality Testing, 2024, 15(16); Identification of components of mulberry and sugarcane molasses polyphenols and the antioxidant activity of their compound, Modern Food Science and Technology, 2023, 39(07)), it shows that exploring the complex components and effects of TCM is still an arduous and long-term task. Unfortunately, there is still a lack of modern medical systematic research on the effects of mulberries on bone formation, marrow production, and joint health, and there are no reports of total polyphenols from mulberries promoting bone formation or preventing osteoporosis. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention utilizes 3D bone formation visualization and quantitative evaluation methods to screen traditional Chinese medicines with evidence-based medical records. For the first time, it has discovered the outstanding efficacy of mulberry extract in promoting bone formation. Using this method, it has tracked and confirmed that total polyphenols from mulberry are the effective component. Based on this, it provides an application of total polyphenol extract from mulberry in the preparation of products that promote bone formation.

[0007] The specific technical solution is as follows:

[0008] Application of a total polyphenol extract from mulberry in the preparation of products that promote bone formation.

[0009] Furthermore, products that promote bone formation can achieve one or more of the following effects:

[0010] (1) Promotes osteoblast proliferation;

[0011] (2) Promotes osteogenic calcification;

[0012] (3) Inhibits the activity of osteosclerosing proteins;

[0013] (4) Promotes bone growth.

[0014] The products that promote bone formation are those that promote fracture healing and growth or combat osteoporosis.

[0015] Furthermore, mulberry total polyphenol extract can be used alone or in combination to prepare products that promote bone formation.

[0016] Furthermore, the total polyphenol content in the mulberry total polyphenol extract ranges from 2% to 90%.

[0017] Furthermore, the active ingredients of the mulberry total polyphenol extract include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, and tannins.

[0018] Furthermore, the phenols and their derivatives in the total polyphenol extract of mulberry mainly include pelargonidin-3-O-chlororutin, protocatechuic acid, cyanidin-3-O-galactoside, 8-isopentenyl naringenin, quercetin, kaempferol, proanthocyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechin, epigallocatechin gallate, and vanillic acid.

[0019] Furthermore, the mulberry total polyphenol extract is a crude mulberry total polyphenol extract obtained by solvent extraction, and a refined mulberry total polyphenol extract and its monomers obtained by refining the crude mulberry total polyphenol extract.

[0020] Furthermore, the solvent is water or ethanol, preferably ethanol.

[0021] Furthermore, the preparation method of the mulberry total polyphenol extract is as follows:

[0022] (1) Dry and pulverize the mulberries to make mulberry powder;

[0023] (2) Dissolve mulberry powder in 70% ethanol, stir thoroughly, heat under ultrasonic assistance, centrifuge and collect the supernatant; then add 70% ethanol to the residue, heat under ultrasonic assistance and centrifuge, collect the supernatant.

[0024] (3) Combine the collected supernatants, concentrate under reduced pressure to obtain concentrated solution;

[0025] (4) Pretreated D101 macroporous adsorption resin is packed into a glass chromatography column. The concentrate is diluted 4 times and then loaded into the glass chromatography column. After adsorption is completed, some impurities are removed by elution with distilled water. Then, the column is eluted sequentially with 10% ethanol solution, 30% ethanol solution and 60% ethanol solution. The eluent is collected. The pretreatment is carried out in accordance with the relevant methods of "Pretreatment Method of Ion Exchange Resin" in the National Standard of the People's Republic of China (GB / T 5476-2013).

[0026] (5) The eluent was concentrated under reduced pressure to remove ethanol, diluted 10 times with water, and spray-dried to obtain mulberry total polyphenol extract.

[0027] Furthermore, the product is one of the following: pharmaceuticals, health foods, functional foods, and food additives; the pharmaceuticals include tablets, capsules, injections, granules, and suspensions; the health foods include tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and alcohol; the functional foods include functional dairy products, bread, and beverages.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. This invention provides the application of mulberry total polyphenol extract in the preparation of products that promote bone formation. Using a 3D visualization method for evaluating bone formation, it was found that mulberry total polyphenol extract significantly promotes osteoblast proliferation and differentiation, exhibiting a dose-response relationship. Further in vitro and in vivo experiments verified that mulberry total polyphenol extract has a bone-promoting effect, consistently showing a clear dose-response relationship. Mechanistic studies show that it can inhibit the activity of the classic target osteosclerosing protein, providing a novel methodology and strategy for developing products that promote bone formation, such as those promoting fracture healing and combating osteoporosis.

[0030] 2. This invention also provides a variety of methods for preparing total polyphenol extracts of mulberry. Among them, the total polyphenol content of the mulberry total polyphenol extract obtained by ethanol extraction and macroporous adsorption resin purification is as high as 634.9 mg gallic acid / g. This provides a practical solution for the application of mulberry total polyphenols in the fields of pharmaceuticals, health foods, and functional foods, which helps to improve product quality and reduce production costs. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 is a standard curve diagram for the determination of total polyphenol content in Example 4.

[0033] Figure 2 is a qualitative composition diagram of the main components of phenolic substances and their derivatives in Example 4;

[0034] Figure 3 is a 3D visualization of the bone formation screening experiment results for different total polyphenol contents in Example 5.

[0035] Figure 4 shows the results of the osteoblast proliferation experiment in Example 6.

[0036] Figure 5 shows the experimental results of promoting osteogenic calcification in Example 7.

[0037] Figure 6 shows the results of the subcutaneous ossification experiment in animals in Example 8.

[0038] Figure 7 shows the results of the osteosclerosing protein activity inhibition experiment in Example 9.

[0039] Figure 8 shows the results of the TCF promoter activation experiment in Example 10. Embodiments of the present invention

[0040] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0041] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0042] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in case of any conflict, the specification containing the definitions shall prevail.

[0043] The materials, reagents, and experimental equipment used in this invention are sourced from the following sources:

[0044] The mulberries were purchased from Shandong Lubao Specialty Agriculture Technology Co., Ltd.

[0045] The cellulase was purchased from Shandong Longkete Enzyme Preparation Co., Ltd.

[0046] Pectinase was purchased from Shandong Longkete Enzyme Preparation Co., Ltd.

[0047] Edible citric acid was purchased from Shandong Yingxuan Industrial Co., Ltd.

[0048] Gallic acid was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0049] The elution and extraction solvents were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0050] All reagents used in the LC-MS were of chromatographic grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0051] The cell culture medium was high-glucose DMEM medium supplemented with 10% FBS and 1% antibiotics;

[0052] The calcium ion probe was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0053] CCK-8 solution was purchased from Wuhan Solarbio Biotechnology Co., Ltd.

[0054] Alizarin Red dye solution was purchased from Shanghai Pronosei Biotechnology Co., Ltd.

[0055] The nude mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0056] The matrix adhesive was purchased from Shaanxi Kangning Biotechnology Group Co., Ltd.

[0057] Luciferase lysis buffer and detection kit were purchased from Beijing TransGen Biotech Co., Ltd.

[0058] MC3T3-E1 cells were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0059] Liquid chromatography-ultra-high resolution mass spectrometry system, model OrbitrapExploris 480;

[0060] Nikon confocal microscope, model AR.

[0061] The D101 macroporous adsorption resin used in the examples was pretreated according to the relevant methods in the "Pretreatment Method of Ion Exchange Resins" in the "National Standard of the People's Republic of China (GB / T 5476-2013)".

[0062] Example 1: Preparation of total polyphenol extract from mulberry by water boiling and alcohol precipitation method

[0063] (1) Dry the mulberries at 60°C and then pulverize them using a multi-functional pulverizer to obtain mulberry powder.

[0064] (2) Take 50g of mulberry powder and dissolve it in 500mL of deionized water to obtain the soaking solution.

[0065] (3) Boil the soaking solution for 2 hours.

[0066] (4) Filter to remove the filter residue and concentrate the filtrate under reduced pressure (temperature: 80℃, speed: 90rpm, vacuum degree: 0.09MPa).

[0067] (5) The concentrate was precipitated with 50% ethanol solution and centrifuged (5000 rpm, 5 min) to remove the supernatant and collect the precipitate.

[0068] (6) Dilute the alcohol precipitate 15 times with deionized water and centrifuge (3000 rpm, 5 min). Collect the supernatant and spray dry (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry total polyphenol extract A.

[0069] Example 2: Preparation of total polyphenol extract from mulberry by ultrasonic enzymatic hydrolysis extraction

[0070] (1) Dry the mulberries at 60°C and then pulverize them using a multi-functional pulverizer to obtain mulberry powder.

[0071] (2) Take 50g of mulberry powder, dissolve it in 450mL of deionized water, add 5g of pectinase and 5g of cellulase, and perform ultrasonic enzymatic hydrolysis extraction. The extraction temperature is 50℃, the extraction time is 10min, and the ultrasonic power is 150W.

[0072] (3) Add 50 mL of anhydrous ethanol to the aqueous extract to form a 10% ethanol solution by volume, and add 5 g of citric acid to form an acidified ethanol solution. Perform ultrasonic extraction at 50 °C for 20 min with an ultrasonic power of 150 W to obtain the ethanol extract.

[0073] (4) The alcohol extract was repeatedly ultrasonically extracted with 10 times the volume of ethyl acetate at an extraction temperature of 40°C and an ultrasonic power of 150W. The extraction was repeated 3 times. The organic phase was discarded and the aqueous phase was retained.

[0074] (5) Dilute the aqueous phase with deionized water 10 times and spray dry (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry total polyphenol extract B.

[0075] Example 3: Preparation of Mulberry Total Polyphenol Extract by Alcohol Extraction and Refining Method

[0076] (1) Dry the mulberries at 60°C and then pulverize them using a multi-functional pulverizer to obtain mulberry powder.

[0077] (2) Take 50g of mulberry powder, dissolve it in 500mL of 70% ethanol, stir thoroughly, and then perform ultrasonic ethanol extraction. The extraction temperature is 60℃, the time is 6h, the ultrasonic power is 150W, centrifuge (5min, 5000rpm), and collect the supernatant. Then, add 500mL of 70% ethanol to the residue, and continue ultrasonic ethanol extraction. The extraction temperature is 60℃, the time is 1h, the ultrasonic power is 150W, centrifuge (5min, 5000rpm), and collect the supernatant. Repeat twice.

[0078] (3) Combine the supernatants from the three times and concentrate them under reduced pressure (temperature: 60℃, rotation speed: 90rpm, vacuum degree: 0.09MPa) to concentrate the volume to one column volume of a macroporous resin column (diameter: 1.6cm, height: 50cm).

[0079] (4) A glass chromatography column with a diameter of 1.6 cm and a height of 50 cm was packed with pretreated D101 macroporous adsorption resin. The concentrated mulberry polyphenol solution diluted 4 times with distilled water was loaded into the glass chromatography column at a flow rate of 1 mL / min at 60 °C. After adsorption was completed, 100 mL of distilled water was used to remove some impurities. Then, 100 mL of 10% ethanol solution, 200 mL of 30% ethanol solution and 300 mL of 60% ethanol solution were used to elute in sequence. Finally, the eluent was collected.

[0080] (5) First, the eluent is concentrated under reduced pressure (temperature: 80℃, speed: 90rpm, vacuum degree: 0.09MPa) to remove ethanol. Then, the eluent concentrate is diluted 10 times with deionized water and spray dried (inlet air temperature: 180℃, needle: 5s / time, peristaltic pump: 30%) to obtain mulberry polyphenol extract C.

[0081] Example 4: Determination of total polyphenol content and identification of components in total polyphenol extracts of mulberry (Examples 1-3).

[0082] 1. Determination of total polyphenol content in mulberry total polyphenol extract in Examples 1-3

[0083] Referring to page 69 of the Pharmacopoeia of the People's Republic of China (2020 Edition, Part I) and the "Determination Method of Total Phenolic Content" in the National Standard of the People's Republic of China (GB / T44349-2024), the total polyphenol content of mulberry total polyphenol extracts A, B, and C prepared in Examples 1-3 was determined.

[0084] (1) Preparation of reference solution

[0085] Take an appropriate amount of gallic acid reference standard, accurately weigh it, and add water to prepare a solution containing 50 μg per 1 mL to obtain the reference standard solution.

[0086] (2) Preparation of standard curve

[0087] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.4 mL of the reference solution into separate 10 mL volumetric flasks. Add 6 mL of water, shake well, then add 0.5 mL of Folin-Ciocalteu solution B, shake well, and add 1.5 mL of 20% sodium carbonate solution over 0.5–8 min. Dilute to the mark with water and shake well. Incubate in a 75°C water bath for 10 min. Using the corresponding reagents as blanks, measure the absorbance at 760 nm using UV-Vis spectrophotometry. Plot a standard curve (see Figure 1) with absorbance as the ordinate and concentration as the abscissa. The regression equation is: Y = 0.0572X + 0.0731, R0 2 =0.9991.

[0088] (3) Weigh 2 mg of the extract accurately and place it in a 2 mL centrifuge tube. Add deionized water to the mark to obtain a diluted solution. Take 1.4 mL of the diluted solution into a 10 mL volumetric flask. Following the method in step (2), starting from "add 6 mL of water", determine the absorbance according to the method. Read the concentration of gallic acid in the test solution from the standard curve. The calculation results are shown in Table 1.

[0089] Table 1. Results of determination of total polyphenol content in mulberry total polyphenol extracts in Examples 1-3

[0090]

[0091] 2. The components in the mulberry total polyphenol extract prepared in Example 3 were identified using liquid chromatography-mass spectrometry (LC-MS).

[0092] (1) Accurately weigh 1 g of total polyphenol extract of mulberry, dissolve it in 50 mL of methanol, and place it in an ultrasonic cleaner for ultrasonic extraction for 30 min;

[0093] (2) After centrifugation (10000 rpm, 10 min), take the supernatant;

[0094] (3) The supernatant was filtered through a 0.22 μm filter membrane to obtain the sample solution to be tested. A C18 column was used at a column temperature of 40 °C, and acidified water and acetonitrile were selected as the mobile phase. The secondary spectrum was captured using an electrospray ionization source (ESI) in positive ion mode.

[0095] (4) Data analysis:

[0096] 4.1 The total polyphenol extract of mulberry was analyzed by LC-MS to obtain the retention time of the chromatographic peaks and mass spectrometry data.

[0097] 4.2 The acquired retention time and mass spectrometry data were compared with the retention time and mass spectrometry information in the MS-Dail spectral library to identify the main polyphenolic components in the extract.

[0098] 4.3 For unknown peaks, structural analysis and identification are performed by combining literature data and mass spectrometry fragment information.

[0099] Experimental Results: LC-MS analysis revealed that the main components of phenolic compounds include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, tannins, and other phenolic substances, as shown in Figure 2. Further structural analysis and identification, combining literature data and mass spectrometry fragment information, identified 16 components of phenols and their derivatives, as detailed in Table 2.

[0100] Table 2. Results of total polyphenol content determination in mulberry total polyphenol extract (Example 3)

[0101]

[0102] Example 5 3D Visual Screening Experiment on the Ability of Total Polyphenol Extracts from Mulberries in Examples 1 - 3 to Promote Osteoblast Differentiation

[0103] (1) Accurately weigh appropriate amounts of the 3 total polyphenol extracts from mulberries obtained in Examples 1, 2, and 3, and dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, which are stored at -20°C for later use.

[0104] (2) Add 500 μL of surfactant to each well of the 3D microwell plate and rinse with cell culture medium. Digest the extracted and cultured MC3T3-E1 cells and count them to obtain a cell suspension with a final concentration of 2.4×10 5 / 100 mL. Slowly pipette the cell suspension along the well wall to the bottom and culture overnight.

[0105] (3) After observing the appearance of 3D cell spheres, replace the original culture medium with a culture medium containing 3 total polyphenol extracts from mulberries with a final concentration of 0.03 mg / mL each; set 4 replicates for each concentration. Use MC3T3-E1 cells cultured in normal medium as the blank control group, use the medium of total polyphenol extract A from mulberries in Example 1 as the Example 1 group, use the medium of total polyphenol extract B from mulberries in Example 2 as the Example 2 group, and use the medium of total polyphenol extract C from mulberries in Example 3 as the Example 3 group.

[0106] (4) On the 3rd day of culture, use a calcium ion fluorescent probe to label calcified nodules and analyze the calcification levels of each group through quantification software. The results are shown in Figure 3.

[0107] As can be seen from Figure 3, compared with the blank control group, the three total polyphenol extracts from mulberries with different contents can significantly enhance osteogenic calcification deposition, and at the same time, there is an obvious dose-effect relationship with the increase in the total polyphenol content.

[0108] Example 6 Experiment on the Promotion of Osteoblast Proliferation by Total Polyphenol Extract C from Mulberries in Example 3

[0109] (1) Accurately weigh appropriate amounts of total polyphenol extract C from mulberries obtained in Example 3 and dissolve them in double-distilled water to prepare 20 mg / mL stock solutions, which are stored at -20°C for later use.

[0110] (2) After diluting the mother liquor to 0.015 mg / mL, 0.03 mg / mL, and 0.06 mg / mL respectively, it was used to culture MC3T3-E1 cells in a 96-well plate for 72 h. Then, a CCK-8 solution with a final concentration of 10% was added, and the OD value in the 96-well plate was measured using an enzyme-labeled instrument at an absorbance of 450 nm. Each concentration was set with 4 replicates. Culturing MC3T3-E1 cells with ordinary medium was used as the blank control group, culturing MC3T3-E1 cells with 0.15 mg / mL extract added was used as Example 3-1 group, culturing MC3T3-E1 cells with 0.03 mg / mL extract added was used as Example 3-2 group, and culturing MC3T3-E1 cells with 0.06 mg / mL extract added was used as Example 3-3 group. The results are shown in Figure 4.

[0111] As can be seen from Figure 4, compared with the blank control group, the total mulberry polyphenol extract in Example 3 can significantly enhance the activity of osteoblasts and promote the proliferation of osteoblasts. Moreover, as the dose increases, the promoting effect gradually increases, showing an obvious dose-effect relationship.

[0112] Example 7 Experiment on the promotion of osteoblast calcification by total mulberry polyphenol extract C

[0113] (1) Accurately weigh an appropriate amount of the total mulberry polyphenol extract C obtained in Example 3, and dissolve it with double-distilled water to prepare a 20 mg / mL mother liquor and store it at -20 °C for later use.

[0114] (2) After diluting the mother liquor to 0.015 mg / mL, 0.03 mg / mL, and 0.06 mg / mL respectively, it was used to culture MC3T3-E1 cells in a 24-well plate for 72 h. Then, a tissue fixing solution was added for alizarin red staining. After 30 min, a 0.1% cetylpyridinium chloride solution was added to each well, and the OD value in the 24-well plate was measured using an enzyme-labeled instrument at an absorbance of 570 nm after 5 min. Each concentration was set with 4 replicates. Culturing MC3T3-E1 cells with ordinary medium was used as the blank control group, culturing MC3T3-E1 cells with 0.015 mg / mL extract added was used as Example 3-1 group, culturing MC3T3-E1 cells with 0.03 mg / mL extract added was used as Example 3-2 group, and culturing MC3T3-E1 cells with 0.06 mg / mL extract added was used as Example 3-3 group. The results are shown in Figure 5.

[0115] As can be seen from Figure 5, compared with the blank control group, the total mulberry polyphenol extract C in Example 3 can significantly enhance osteoblast calcification, and as the dose increases, this promoting effect gradually increases, showing an obvious dose-effect relationship.

[0116] Example 8 Experiment on the promotion of subcutaneous osteogenesis in animals by total mulberry polyphenol extract

[0117] (1) Accurately weigh an appropriate amount of mulberry total polyphenol extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 50 mg / mL mother liquor, and store it at 4°C for later use.

[0118] (2) Subcutaneous bone injection experiment:

[0119] Nude mice were randomly divided into four groups of five mice each. MC3T3-E1 cells were cultured and expanded to a cell count of 1×10⁻⁶. 7 The cells were then digested and prepared for subcutaneous bone injection. 1 × 10⁻⁶ cells were injected into the same location in each nude mouse. 6 / 100μL of MC3T3-E1 suspension and 50μL of matrix gel. On the first day after the injection, nude mice in each group were administered oral gavage. The blank control group, low-dose group, medium-dose group and high-dose group were administered physiological saline, 2.5mg, 5mg and 10mg of total polyphenol extract of mulberry C from Example 3, respectively, once a day until the end of the 7-day experiment.

[0120] (3) Methods for measuring bone tissue volume:

[0121] The nude mice were observed daily, and changes in subcutaneous bone tissue volume were recorded using calipers. The recording principle was: measure the maximum long axis (L) and short axis (H) of the tissue, according to V = 1 / 2 × L. 2 The volume of the tissue was calculated and quantified using ×H, and the results are shown in Figure 6.

[0122] As shown in Figure 6, compared with the blank control group, the total polyphenol extract of mulberry in Example 3 significantly enhanced subcutaneous bone growth. With increasing oral dosage, the volume of bone tissue gradually increased, exhibiting a dose-response relationship.

[0123] Example 9: Experiment on the inhibition of osteoporosis treatment target osteosclerosin protein activity by the total polyphenol extract of mulberry in Example 3.

[0124] (1) Accurately weigh an appropriate amount of mulberry total polyphenol extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL mother liquor, and store it at -20℃ for later use.

[0125] (2) The mother liquor was diluted to 0.015 mg / mL, 0.03 mg / mL and 0.06 mg / mL respectively, and then used to culture MC3T3-E1 cells in 6-well plates for 72 h. MC3T3-E1 cells cultured with the culture medium were used as blank control group. MC3T3-E1 cells cultured with 0.015 mg / mL extract were used as Example 3-1. MC3T3-E1 cells cultured with 0.03 mg / mL extract were used as Example 3-2. MC3T3-E1 cells cultured with 0.06 mg / mL extract were used as Example 3-3.

[0126] (3) Total cell protein was obtained using RIPA protein lysis buffer and protein concentration was quantified using the BCA method. The quantitative standard was 30 μg of protein per sample.

[0127] (4) After SDS-PAGE electrophoresis of the protein, the protein was transferred to a PVDF membrane. Non-specific binding sites were blocked with 5% BSA solution. After overnight incubation with 1:1000 osteoscleroprotein primary antibody, binding was performed with 1:5000 horseradish peroxidase-labeled goat anti-rabbit secondary antibody. Finally, ECL colorimetric analysis was used to observe the inhibitory effect of mulberry total polyphenol extract on osteoblast osteoscleroprotein activity. The results are shown in Figure 7.

[0128] As shown in Figure 7, compared with the blank control group, the total polyphenol extract of mulberry in Example 3 can significantly inhibit the expression of osteosclerosingin protein, and the inhibitory effect gradually increases with increasing dose.

[0129] Example 10: Activation experiment of the TCF promoter downstream of the osteoporosis treatment target by mulberry total polyphenol extract C in Example 3.

[0130] (1) Preparation of pCMV3-TCF-LUC vector:

[0131] A fragment carrying the fusion of the TCF and LUC genes was amplified using polymerase chain reaction (PCR). Unbroken polymerase chains were added to the ends of the sense and antisense strands. After amplification and purification, the fragment was ligated into the pCMV3 vector, and the target vector was obtained through prokaryotic transformation using *E. coli*. The target vector, along with psPAX2 and pMD2.G vectors at a ratio of 4:3:1, was used to prepare retroviruses in 293T cells via lipo2000 liposomes. The collected viruses were concentrated for infecting MC3T3-E1 cells.

[0132] (2) Construction of MC3T3-TCF-LUC cell line:

[0133] The concentrated virus was added to MC3T3-E1 cells at a ratio of 1:1000 and infected for 48 hours. After discarding the supernatant containing the virus, the cells were screened using cell culture medium containing 2.5 μg / mL puromycin. After 72 hours of screening, the remaining surviving cells were collected and cultured to obtain a stable MC3T3-TCF-LUC cell line.

[0134] (3) Experiment on the activation of luciferase in MC3T3-TCF-LUC cells by total polyphenol extract of mulberry:

[0135] Accurately weigh an appropriate amount of mulberry total polyphenol extract C obtained in Example 3, dissolve it in double-distilled water to prepare a 20 mg / mL stock solution, and store it at -20℃ for later use. Dilute the stock solution to 0.015 mg / mL, 0.03 mg / mL, and 0.06 mg / mL, respectively, and culture MC3T3-E1 cells in 96-well plates for 24 h. MC3T3-E1 cells cultured with double-distilled water served as the blank control group. MC3T3-E1 cells cultured with 0.015 mg / mL extract were designated as Example 3-1, MC3T3-E1 cells cultured with 0.03 mg / mL extract were designated as Example 3-2, and MC3T3-E1 cells cultured with 0.06 mg / mL extract were designated as Example 3-3.

[0136] After adding cell lysis buffer, the activation of TCF by the total polyphenol extract of mulberry was tested using a luciferase assay kit. The results are shown in Figure 8.

[0137] As shown in Figure 8, compared with the blank control group, the total polyphenol extract of mulberry can significantly activate the TCF promoter, and the activation effect gradually increases with the increase of total polyphenol content.

Claims

1. The application of a total polyphenol extract of mulberry in the preparation of products that promote bone formation.

2. The application as described in claim 1, characterized in that, The bone formation promoting product can achieve one or more of the following effects: (1) Promotes osteoblast proliferation; (2) Promotes osteogenic calcification; (3) Inhibits the activity of osteosclerosing proteins; (4) Promotes bone growth.

3. The application as described in claim 2, characterized in that, The products that promote bone formation are those that promote fracture healing and growth or combat osteoporosis.

4. The application as described in claim 1, characterized in that, The total polyphenol extract of mulberry can be used alone or in combination to prepare products that promote bone formation.

5. The application as described in claim 1, characterized in that, The total polyphenol content in the mulberry total polyphenol extract is 2% to 90%.

6. The application as described in claim 1, characterized in that, The active ingredients of the mulberry total polyphenol extract include phenolic acids, polyphenol derivatives, flavonoids, isoflavones, and tannins.

7. The application as described in claim 6, characterized in that, The phenols and their derivatives in the total polyphenol extract of mulberry mainly include pelargonidin-3-O-chlororutin, protocatechuic acid, cyanidin-3-O-galactoside, 8-isopentenyl naringenin, quercetin, kaempferol, proanthocyanidin A2, luteolin, caffeic acid, chlorogenic acid, resveratrol, myricetin, gallic acid, catechin, epigallocatechin gallate, and vanillic acid.

8. The application as described in claim 1, characterized in that, The mulberry total polyphenol extract is a crude mulberry total polyphenol extract obtained by solvent extraction, and a refined mulberry total polyphenol extract and its monomers obtained by refining the crude mulberry total polyphenol extract.

9. The application as described in claim 1, characterized in that, The preparation method of the total polyphenol extract of mulberry is as follows: (1) Dry and pulverize the mulberries to make mulberry powder; (2) Dissolve mulberry powder in 70% ethanol, stir thoroughly, heat under ultrasonic assistance, centrifuge and collect the supernatant; then add 70% ethanol to the residue, heat under ultrasonic assistance and centrifuge, collect the supernatant. (3) Combine the collected supernatants, concentrate under reduced pressure to obtain concentrated solution; (4) Pretreated D101 macroporous adsorption resin is packed into a glass chromatography column. The concentrate is diluted 4 times and then loaded into the glass chromatography column. After adsorption is completed, some impurities are removed by elution with distilled water. Then, the column is eluted sequentially with 10% ethanol solution, 30% ethanol solution and 60% ethanol solution. The eluent is collected. The pretreatment is carried out in accordance with the relevant methods of "Pretreatment Method of Ion Exchange Resin" in the National Standard of the People's Republic of China (GB / T 5476-2013). (5) The eluent was concentrated under reduced pressure to remove ethanol, diluted 10 times with water, and spray-dried to obtain mulberry total polyphenol extract.

10. The application as described in claim 1, characterized in that, The product is one or more of the following: pharmaceuticals, health foods, functional foods, and food additives; the pharmaceuticals include one or more of the following: tablets, capsules, injections, granules, and suspensions; the health foods include one or more of the following: tablets, hard capsules, soft capsules, oral liquids, milk powder, biscuits, candies, beverages, and alcohol; the functional foods include one or more of the following: functional dairy products, bread, and beverages.

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