Application of 6-C-(E-styryl)-naringenin in preparation of product for preventing and improving osteoporosis

Abstract

The invention discloses an application of 6-CEPN in preparation of a product for preventing and improving osteoporosis. According to the invention, ovariectomized mice are fed with two doses of 6-CEPN, which discovers that the 6-CEPN can obviously inhibit abnormal weight increase of the mice caused by estrogen decrease and can also improve bone microstructure of the ovariectomized mice; the bone trabecula network structure is adjusted by increasing the bone trabecula connection density, the bone trabecula number and the bone trabecula thickness and reducing the separation degree of the bone trabecula, meanwhile, 6-CEPN can further increase the bone density and the bone volume fraction of ovariectomized mice, increase the rigidity and deflection of bones and improve the bone strength, the effect of high-dose 6-CEPN is larger than that of low-dose 6-CEPN, and the effect of high-dose 6-CEPN is better than that of low-dose 6-CEPN. Therefore, the composition has a dose-dependent effect.

Description

Technical Field

[0001] This invention belongs to the field of developing new uses for food molecular substances, specifically involving the application of 6-C-(E-styryl)-naringenin in the preparation of products for preventing and improving osteoporosis. Background Technology

[0002] Osteoporosis is a chronic degenerative bone disease characterized by decreased overall bone mass, deterioration of bone microstructure, increased bone fragility, and a higher risk of fractures. Postmenopausal osteoporosis is caused by a decline in estrogen levels, leading to reduced inhibition of osteoclast activity and a greater bone resorption than remodeling activity, resulting in decreased bone quality. Statistics show that approximately one-third of postmenopausal women aged 50-70 suffer from osteoporosis. Over 80% of women over 80 years of age have osteoporosis. The probability of developing osteoporosis in postmenopausal middle-aged and elderly women is significantly higher than in men; therefore, the treatment of postmenopausal osteoporosis is of paramount importance.

[0003] Skeleton is one of the most important organs in the human body, playing multiple roles in support, protection, movement, hematopoiesis, storage, and metabolism. Various cell types exist within the skeleton, among which osteoblasts and osteoclasts are closely related to bone metabolism. Bone homeostasis is the dynamic balance between osteoblast-mediated bone remodeling and osteoclast-mediated bone resorption under physiological conditions. When bone homeostasis is disrupted, bone resorption activity exceeds bone remodeling activity, leading to osteoporosis. Therefore, enhancing osteoblast activity or inhibiting osteoclast activity are key targets for treating osteoporosis.

[0004] There are many drugs used clinically to treat osteoporosis, which are generally divided into two main categories: bone resorption inhibitors and bone remodeling promoters. Bisphosphonates and estrogen drugs are commonly used bone resorption inhibitors. The former can inhibit the differentiation of osteoclasts, but oral administration can cause gastrointestinal abnormalities such as nausea and vomiting. The latter, when used at high doses, can stimulate uterine cells and lead to uterine lesions. Therefore, seeking safer and more harmless methods to improve and treat osteoporosis has become the focus of research. Summary of the Invention

[0005] To address the aforementioned problems, the purpose of this invention is to provide a safe and harmless substance that can prevent, improve, and treat osteoporosis, thereby solving the problems of high cost and side effects of existing osteoporosis treatment drugs.

[0006] To achieve the above objectives, the present invention first provides the use of 6-C-(E-styryl)naringenin or a composition containing 6-C-(E-styryl)naringenin in the preparation of products for preventing and improving osteoporosis.

[0007] In one embodiment of the present invention, the osteoporosis includes osteoporosis caused by a decline in estrogen levels, including senile osteoporosis and postmenopausal osteoporosis, preferably postmenopausal osteoporosis.

[0008] In one embodiment of the present invention, the improvement of osteoporosis includes improving bone microstructure and increasing bone strength.

[0009] In one embodiment of the present invention, the improvement of bone microstructure includes improving the trabecular bone network structure.

[0010] In one embodiment of the present invention, the improvement of the trabecular network structure includes increasing bone density and bone volume fraction, increasing trabecular connection density, trabecular number and trabecular thickness, and reducing trabecular separation.

[0011] In one embodiment of the present invention, the increase in bone strength includes increasing the stiffness and deflection of the muscular bones.

[0012] In one embodiment of the present invention, the product includes functional food, pharmaceutical, or health food.

[0013] In one embodiment of the present invention, the functional food includes at least one of functional beverages, functional powders, functional biscuits, and functional capsules.

[0014] In one embodiment of the invention, the medicine includes a drug for the prevention and treatment of osteoporosis.

[0015] In one embodiment of the present invention, the health food includes a health food for increasing bone density.

[0016] The present invention also discloses a health food composition for increasing bone density, the composition comprising 6-C-(E-styryl)naringenin.

[0017] The present invention also discloses a pharmaceutical composition for the prevention and treatment of osteoporosis, the composition comprising 6-C-(E-styryl)naringenin.

[0018] In one embodiment of the present invention, the composition further includes at least one of corn starch, maltodextrin, soy protein, whey protein, calcium carbonate, inulin, fructooligosaccharides, and resistant dextrin.

[0019] Beneficial effects: 1. 6-C-(E-styryl)-naringenin (6-CEPN) is a small molecule substance found during the roasting of beef. It is non-toxic and does not produce toxic side effects. It can significantly improve abnormal weight gain caused by estrogen deficiency and reduced fat metabolism rate.

[0020] 2. This invention demonstrates through animal experiments that 6-C-(E-styryl)-naringenin helps to improve abnormal weight gain in mice caused by estrogen deficiency, improve bone density and trabecular meshwork, improve bone microstructure in ovariectomized mice, inhibit osteoclast activity, and balance bone metabolism, and has a dose-dependent effect.

[0021] 3. 6-C-(E-styryl)-naringenin has a balancing regulatory effect on bone metabolism. Serological tests show that it can alleviate abnormal changes in bone metabolism indicators caused by osteoporosis, especially significantly inhibiting osteoclast activity. TRAP staining shows that 6-C-(E-styryl)-naringenin can effectively inhibit the abnormal increase in the number of osteoclasts, and at the same time, the levels of osteoclast markers (such as TRACP-5b) in serum are also regulated. This indicates that its core mechanism of action lies in effectively inhibiting excessive bone resorption, thereby reversing the negative balance of bone metabolism and maintaining normal bone metabolism.

[0022] 4. 6-C-(E-styryl)-naringenin not only improves the microstructure of bone but also enhances its macroscopic function. Biomechanical tests have shown that intervention with resistant dextrin significantly improves the stiffness and deflection of the mouse femur, indicating that it can effectively improve bone strength and toughness and reduce the risk of fractures. This has crucial practical significance for the clinical treatment of osteoporosis. Attached Figure Description

[0023] Figure 1 The molecular structure diagram of 6-C-(E-styryl)-naringenin.

[0024] Figure 2 This is a schematic diagram of an animal experiment.

[0025] Figure 3 Uterine index and serum estradiol levels were measured in mice in different groups after 10 weeks of gavage administration of the test substance. Data are expressed as mean ± SD. One-way ANOVA was used, followed by Tukey's test to assess statistical significance. Different letters indicate significant differences between groups.

[0026] Figure 4 The effect of different test substances administered via gavage on body weight gain in mice. Data are presented as mean ± SD. One-way ANOVA was used, followed by Tukey's test to assess statistical significance. Different letters indicate significant differences between groups.

[0027] Figure 5 Visceral indices of mice in different groups after 10 weeks of gavage administration of the test substance. Data are expressed as mean ± SD. One-way ANOVA was used, followed by Tukey's test to assess statistical significance. Different letters indicate significant differences between groups.

[0028] Figure 6 Micro-CT images of the distal femoral trabecular bone structure in mice from different groups after 10 weeks of gavage administration of the test substance.

[0029] Figure 7 Images of bone microstructure in mice from different groups after 10 weeks of gavage administration of the test substance are shown. A is an HE staining image of the trabecular bone structure in the distal femur, and B is a TRAP staining image of the trabecular bone structure in the distal femur.

[0030] Figure 8 Bone mineral density (BMD), bone volume fraction (BV / TV), trabecular connectivity (COnn.D), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.S) were measured in different groups of mice after 10 weeks of gavage administration of the test substance. Data are expressed as mean ± SD. One-way ANOVA was used, followed by Tukey's test to assess statistical significance. Different letters represent significant differences between groups.

[0031] Figure 9 The concentrations of BGP, BALP, Cath-K, and TRACP in the serum of mice in different groups after 10 weeks of gavage administration of the test substance were recorded. Data are expressed as mean ± SD. One-way ANOVA was performed, followed by Tukey's test to assess statistical significance. Different letters indicate significant differences between groups.

[0032] Figure 10 The stiffness and deflection of the skeletons of mice in different groups were measured after 10 weeks of gavage administration of the test substance. Data are expressed as mean ± SD. One-way ANOVA was used, followed by Tukey's test to assess statistical significance. Different letters represent significant differences between groups.

[0033] Figure 11 The flowchart shows the animal experiment of inducing mature BMDM osteoclasts with 50 ng / mL LM-CSF and 100 ng / mL RANKL. The concentrations of naringenin were 10 and 50 μmol / L, and the concentrations of 6-C-(E-styryl)-naringenin were 10 and 50 μmol / L, respectively. The culture medium was changed every 2 days, and TRAP staining was performed on day 6.

[0034] Figure 12 for Figure 11 The corresponding TRAP staining image from the experiment, scale bar = 100 μm.

[0035] Figure 13 Quantitative polymerase chain reaction analysis of relevant genes in mature osteoclasts. Detailed Implementation

[0036] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0037] I. Reagents The mice used in this invention were 8-week-old female C57BL / 6J mice, SPF grade, weighing 18-20 g, purchased from Spefair (Suzhou) Laboratory Animal Technology Co., Ltd.; the preparation of 6-C-(E-styrylethenyl)-naringenin was as described in the literature (Kang Q, Gong J, Wang M, Wang Q, Chen F, Cheng KW. 6-C-(E-Phenylethenyl)Naringenin Attenuates the Stemness of Hepatocellular Carcinoma Cells by Suppressing Wnt / β-Catenin Signaling. J Agric Food Chem. 2019 Dec). 18;67(50):13939-13947); The ELISA kit for E2 was purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd.; the ELISA kits for BALP, BGP, TRAP, and CTSK were purchased from Elite Biotechnology Co., Ltd.; α-amylase (high-temperature amylase) and amylase were purchased from Shanghai Myriel Biotechnology Co., Ltd.; the hematoxylin-eosin (H&E) high-definition constant staining kit, environmentally friendly dewaxing and clearing solution, TRAP staining solution kit, hematoxylin staining solution, differentiation solution, and blue return solution were purchased from Servicebio; the immunohistochemistry pen was purchased from Vector; M-CSF and RANKL were purchased from MCE; α-MEM medium was purchased from Solarbio; TRAP staining agent was purchased from Servicebio; and PCR primers for TRAP, CTSK, Nfact-1, and MMP-9 were purchased from Shanghai Sangon Biotech.

[0038] II. Experimental Methods 1. Mouse model construction This experiment was approved by the Animal Ethics Committee of Jiangnan University, with ethics number JN.No20250113C100103

[006] . Forty SPF-grade female C57BL / 6J mice were housed in an environment with a temperature of 23±2℃, humidity of 50±10%, and a 12h daytime and 12h darktime cycle, with free access to food and water during the study period. After one week of acclimatization, the mice were randomly divided into a sham-operated group (Sham group, n=10) and a surgical group (n=30). After anesthesia, the surgical group mice underwent bilateral ovariectomy, while the sham-operated group mice had a small amount of fat removed from the vicinity of both ovaries, and 3-4 drops of sodium penicillin were applied to the wounds, which were then sutured in two layers. Postoperatively, sodium penicillin was injected for three consecutive days to prevent infection.

[0039] Three days after surgery, the mice in the surgical group were divided into two groups according to their body weight: the model control group (OVX) and the low-dose 6-CEPN group (10 mg / kg). -1 ·bw -1 ), high-dose 6-CEPN group (20 mg·kg) -1 ·bw -1 Each group consisted of 10 animals. The Sham and OVX groups were administered physiological saline by gavage, while the remaining groups were administered the corresponding dose of the test substance, such as... Figure 2 As shown. Gavage was administered once daily for 10 consecutive weeks, with weight measured weekly.

[0040] After the 10-week experiment, mice were weighed, blood was collected from their eyeballs, and serum was obtained by centrifugation at 5000×g for 15 min at 4℃. This serum was then stored at -80℃ to avoid repeated freeze-thaw cycles. The right femur of the mice was removed and fixed in 4% paraformaldehyde. The right tibia of the mice was removed, wrapped in gauze moistened with physiological saline, and stored at -20℃. The remaining femurs and tibias were placed in liquid nitrogen and stored at -80℃. Organs such as the liver, kidneys, spleen, and uterus were removed, weighed, and organ indices were calculated.

[0041] 2. Micro-CT scan The distal femur of the right side was scanned using Mirco-CT. The scanning conditions were: voltage 90 kV, current 88 μA, FOV 86 μm, high-resolution scanning mode, and scanning time 4 min. The image and data were analyzed using the instrument's built-in software.

[0042] 3. Serum-related index measurement After the serum was thawed, the levels of E2, BALP, TRACP-5b, BGP, and CTSK in mouse serum were determined using an ELISA kit according to the instructions.

[0043] 4. Measurement of bone biomechanical properties The biomechanical properties of the right tibia of mice were determined using a physical property analyzer. The maximum load on the right tibia was determined by a three-point bending test. A shear probe was selected with a cross-plate spacing of 16 cm. The probe was located directly above the middle of the tibia and moved at a speed of 1 mm / s.

[0044] 5. Bone tissue morphology analysis Hematoxylin-eosin (HE) staining for bone microstructure observation: The right femur was fixed in 4% paraformaldehyde solution, decalcified with 8% EDTA for two weeks, embedded in paraffin, and cut into thin sections of approximately 2 μm. These paraffin sections were baked at 60℃ for 60 min. After baking, staining was performed using the following steps: immersion twice in environmentally friendly dewaxing solution for 20 min each time, followed by treatment with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 min each, and then rinsing with tap water. Then, immersion in hematoxylin staining solution for 3 min, followed by rinsing with tap water. Differentiation was performed using differentiation solution, followed by rinsing with tap water, blueing with blueing solution, and rinsing with running water. The sections were dehydrated in 95% ethanol for 1 min, stained in eosin staining solution for 15 s, and then treated with 75% ethanol, 95% ethanol and anhydrous ethanol for 2 min in sequence. Finally, they were cleared with xylene for 2 min, and the sections were mounted with neutral resin and air-dried.

[0045] Osteoclasts were observed using tartrate-resistant acid phosphatase (TRAP) staining. The staining steps are as follows: 5.1 Preparation of incubation solution: Mix TRAP staining solution B and TRAP staining solution C in equal proportions and prepare immediately. Label this solution B for later use. Dissolve 20 mg of TRAP staining solution D in 1 mL of TRAP staining solution E and label this solution C for later use (solution C needs to be prepared at least 3 days in advance).

[0046] Mix 18 mL of TRAP staining solution A, 1 mL of solution B, and 1 mL of solution C, then add 0.282 g of TRAP staining solution F. After thorough dissolution and filtration, the TRAP incubation solution is ready for use.

[0047] 5.2 TRAP staining: The slides were placed twice in environmentally friendly dewaxing and clearing solution for 20 min each time, followed by treatment with anhydrous ethanol, 95% ethanol, and 75% ethanol for 5 min each, and then washed three times with distilled water. Next, an immunohistochemical pen was used to circle the slides, and the slides were then placed in a humidified chamber and incubated with distilled water at 37°C for 2 h. After incubation, the distilled water was discarded, filtered TRAP incubation solution was added, and the slides were incubated at 37°C in the dark for 20 min. The incubation solution was discarded, and the slides were washed with water. The nuclei were counterstained with hematoxylin for 15 s. The slides were then dehydrated with 75% ethanol, 95% ethanol, and anhydrous ethanol for 2 min each time, and finally cleared with xylene for 2 min. The slides were then mounted with neutral resin. The staining results showed that the cytoplasm of osteoclasts was red, while the nuclei were blue.

[0048] 6. Primary osteoclast culture Bone marrow cells were extracted from the femur and tibia of 8-week-old C57BL / 6J mice for in vitro culture. The isolated cells were resuspended in α-MEM supplemented with 50 ng / mL LM-CSF and seeded at the optimal concentration. After 5 to 7 days of culture, numerous round mononuclear cells differentiated into short spindle-shaped adherent cells, characterized by bone marrow-derived macrophages (BMDM). Subsequently, the culture medium was replaced with α-MEM containing 50 ng / mL LM-CSF and 100 ng / mL RANKL. The culture medium was replaced every two days, and the culture time was extended by 5–7 days to promote osteoclast maturation.

[0049] 7. TRAP staining Osteoclasts were fixed with 4% paraformaldehyde solution and stained with TRAP according to the manufacturer's protocol. The stained osteoclasts were then observed under an inverted fluorescence microscope.

[0050] 8. Quantitative polymerase chain reaction (qRT-PCR) analysis RNA was extracted using Trizol reagent (Invitgen), and then converted to cDNA using a reverse transcription kit (Vazyme, R333-01). The sequences of the primers used are detailed in Table 1. (The last sentence appears to be incomplete and possibly refers to a separate process.) -ΔΔCt The expression level of β-actin was determined by a method.

[0051] Table 1. PCR primer sequence information used in the experiment: TRACP, CTSK, Nfact-1, and MMP-9.

[0052] Example 1 Effects of 6-CEPN on toxicity and abnormal weight gain in ovariectomized mice.

[0053] Ovariectomy leads to a rapid decrease in estrogen levels in mice; therefore, a decrease in serum estradiol levels can serve as a marker of successful model establishment. Figure 3 It was found that, compared with the Sham group, the serum estradiol level and uterine index of mice in the OVX group were significantly lower than those in the Sham group, indicating successful model establishment. Meanwhile, compared with the Sham group, the mice in the OVX group showed greater weight gain. This is because estrogen deficiency easily leads to fat accumulation in the abdomen, and 6-CEPN intervention can alleviate this weight gain. Figure 4 The liver, spleen, and kidneys are vital internal organs. Regardless of the disease being treated, the side effects of medication on these organs should be kept within a reasonable range to avoid causing further harm to the body. Figure 5 The organ index can indicate that the intervention substances used have low toxicity and high safety in mice.

[0054] Example 2 Effects of 6-CEPN on bone microstructure in ovariectomized mice 3D image of bone microstructure in the distal femur of a mouse. Figure 6 As shown, the trabecular meshwork of mice in the Sham group was intact and tightly connected, while the number of trabeculae in mice in the OVX group was reduced and the meshwork was severely damaged. After 6-CEPN intervention, the bone microstructure of the trabeculae was improved and the meshwork was partially restored, with high-dose 6-CEPN showing a more significant effect. Micro-CT was used to analyze the femoral microstructure of mice in different groups, and the results were as follows. Figure 8 The data shown indicate that, compared with the Sham group, the OVX group mice exhibited significant changes in bone mineral density (BMD), bone volume fraction (BV / TV), trabecular connectivity (COnn.D), trabecular number (Tb.N), trabecular thickness (Tb.Tn), and trabecular separation (Tb.Sp). After feeding with 6-CEPN, compared with the OVX group, all parameters except for Tb.Sp decreased, indicating that 6-CEPN has a dose-dependent effect on improving trabecular bone in ovariectomized mice.

[0055] HE staining can further observe changes in the microstructure of the distal femur in mice, such as... Figure 7 As shown in Figure A, compared with Sham, the trabecular meshwork of bone in the OVX group mice was disrupted, and fat vacuoles increased. After 6-CEPN intervention, the trabecular meshwork of bone in the mice was improved, and the improvement effect was more significant with high-dose 6-CEPN. Meanwhile, TRAP staining could be used to observe the number of osteoclasts; osteoclasts were stained wine red (…). Figure 7 B). Following oophorectomy, the number of osteoclasts significantly increased. These changes were alleviated in the 6-CEPN group, indicating that 6-CEPN intervention could inhibit the increase in osteoclast numbers in a dose-dependent manner.

[0056] The levels of bone metabolism-related indicators in blood and urine vary to different degrees depending on age or the individual's bone metabolic disease. Common bone metabolism-related indicators include alkaline phosphatase (ALP), osteocalcin (BGP), tartrate-resistant acid phosphatase-5b (TRACP-5b), and tissue protein K (CTSK). ALP and BGP are substances secreted during osteoblast differentiation and mineralization, primarily regulating bone remodeling; their levels reflect osteoblast activity. TRACP-5b, CTSK, and other osteoclast-related markers are mainly used to regulate bone resorption and verify osteoclast activity. Figure 9 The levels of bone metabolism markers in the serum of different groups of mice were shown. Figure 9 It can be seen that 6-CEPN can alleviate the abnormal changes in various indicators caused by osteoporosis, indicating that 6-CEPN treatment has a balancing effect on bone metabolism in the experimental group. Among them, the high-dose 6-CEPN is more effective.

[0057] Example 3 Effects of 6-CEPN on bone strength in ovariectomized mice The stiffness and deflection of bones can reflect bone strength, such as Figure 10 As shown, compared with the Sham group, the bone stiffness and deflection of OVX mice were significantly reduced, and both were improved to some extent after 6-CEPN intervention. These results indicate that 6-CEPN can improve the damaged bone microstructure and increase bone strength. The effect of high-dose 6-CEPN is more significant, indicating that it has a dose-dependent effect.

[0058] Example 4 Effects of 6-CEPN on osteoclastogenesis in ovariectomized mice Comparative Example 1 Effect of naringenin on osteoclastogenesis in ovariectomized mice 6-CEPN is known to be a derivative of naringenin, with a styrene group attached to naringenin. However, the effect of this group on the action of naringenin is unknown. Animal experiments show that both 6-CEPN and naringenin can improve postmenopausal osteoporosis, and their effects on inhibiting osteoclasts are more significant. To investigate the effects of 6-CEPN and naringenin on osteoclastogenesis, we stimulated bone marrow-derived macrophages (BMDM) in vitro with RANKL and M-CSF to induce osteoclast differentiation. TRAP staining was used to observe the number of osteoclasts, which were stained wine red. TRAP staining results showed that both 6-CEPN and naringenin inhibited the generation of mature osteoclasts, with 6-CEPN showing a better inhibitory effect on osteoclast maturation. Analysis of the number and area of ​​osteoclasts further confirmed these findings. Figure 12Simultaneously, under 6-CEPN intervention, the mRNA expression of osteoclastogenesis-related genes such as CTSK, TRACP, MMP9, and NFATC1 was significantly inhibited. Figure 13 ).

[0059] The experimental results show that 6-CEPN effectively inhibited osteoclast formation and maturation, and the inhibitory effect was higher than that of naringenin. It is speculated that the increased inhibitory effect may be due to the new structure formed after the reaction of phenylacetaldehyde and naringenin to generate 6-CEPN, which improves the bioavailability of naringenin. Studies have shown that azoxifene is a compound containing a styrene structure. Population studies have shown that azoxifene has no significant effect on reducing the risk of non-vertebral fractures in women with osteoporosis (Arzoxifene for Prevention of Fractures and Invasive Breast Cancer in Postmenopausal Women). At the same time, a metabolomics study related to the efficacy of the anti-osteoporosis drug (zoledronic acid) showed that the sulfate metabolite of 4-vinylphenol (4-hydroxystyrene) was negatively correlated with the increase in vertebral bone mineral density (BMD) induced by zoledronic acid (Identifying therapeutic biomarkers of zoledronic acid by metabolomics). This confirms that grafting a styrene group does not necessarily improve the effect of osteoporosis.

[0060] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. Use of 6-C-(E-styryl) naringenin or a composition containing 6-C-(E-styryl) naringenin in the preparation of a product for preventing and improving osteoporosis.

2. Use according to claim 1, characterized in that, The osteoporosis includes osteoporosis caused by a decrease in estrogen levels, including senile osteoporosis and postmenopausal osteoporosis.

3. Use according to claim 2, characterized in that, The improvement of osteoporosis includes improvement of bone microstructure and increase of bone strength.

4. Use according to claim 3, characterized in that, The improvement of bone microstructure includes improvement of trabecular network structure.

5. Use according to claim 4, characterized in that, The improvement of trabecular network structure includes increase of bone density and bone volume fraction, increase of trabecular connectivity density, trabecular number and trabecular thickness, and decrease of trabecular separation.

6. Use according to claim 3, characterized in that, The increase of bone strength includes increase of rigidity and flexibility of the bone.

7. The use according to claim 1, characterized in that, The product includes a functional food, a pharmaceutical product or a health food.

8. Use according to claim 7, characterized in that The functional food includes at least one of a functional beverage, a functional granule, a functional biscuit and a functional capsule, the pharmaceutical product includes a drug for preventing and treating osteoporosis, and the health food includes a health food for increasing bone density.

9. A pharmaceutical composition for preventing and treating osteoporosis, characterized by, The composition contains 6-C-(E-styryl) naringenin.

10. The use according to claim 1, characterized in that, The composition further includes at least one of corn starch, malt dextrin, soybean protein, whey protein, calcium carbonate, inulin and fructo-oligosaccharide, and resistant dextrin.

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