Application of Morkotin A in the preparation of pharmaceuticals, health foods, or functional foods

Abstract

This invention discloses the application of Morkotin A (quercetin-3-O-rutin-7-O-glucoside) in the preparation of pharmaceuticals, health foods, or functional foods, belonging to the field of biomedical technology. Animal experiments have demonstrated that Morkotin A can significantly increase bone mineral density in ovariectomized osteoporotic mice, improve bone microstructure, and regulate calcium and phosphorus metabolism and bone turnover marker levels. Simultaneously, in vitro cell experiments have confirmed that Morkotin A can significantly promote osteoblast differentiation and inhibit osteoclast formation. The Morkotin A used in this invention is derived from the medicinal and edible herb, Lycium barbarum leaf, and has high safety. Both in vitro and in vivo experiments have confirmed that Morkotin A promotes bone formation and inhibits bone resorption, making it suitable for development as a drug for the prevention and treatment of osteoporosis, or as a health food or functional food that helps improve bone mineral density.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically relates to the application of Morkotin A in the preparation of pharmaceuticals, health foods or functional foods. Background Technology

[0002] Osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass, bone microarchitectural deterioration, and increased bone fragility. It is prevalent in postmenopausal women and the elderly, easily leading to fractures and severely impacting quality of life. Current clinical treatments primarily rely on bisphosphonates, estrogens, or selective estrogen receptor modulators, which present challenges such as gastrointestinal irritation and limited safety with long-term use. Naturally derived, low-toxicity, and highly effective anti-osteoporosis active ingredients have become a hot topic in new drug development.

[0003] Morkotin A, also known as quercetin-3-O-rutinoside-7-O-glucoside, has the English name Quercetin 3-O-rutinoside 7-O-glucoside, CAS number 30311-61-6, and molecular formula C. 33 H 40 O 21 Morkotin A, with a molecular weight of 772.66, is a flavonoid compound isolated from wolfberry leaves. Currently, there are no reports on its use in the prevention and treatment of osteoporosis, nor are its pharmacological effects and mechanisms of action against osteoporosis publicly disclosed. Therefore, clarifying the application of Morkotin A in the treatment of osteoporosis is of great significance for the development of innovative drugs based on Morkotin A and wolfberry leaves. Summary of the Invention

[0004] In view of this, the present invention provides the application of Morkotin A in the preparation of pharmaceuticals, health foods, or functional foods. Animal experiments have demonstrated that Morkotin A can significantly increase bone mineral density in ovariectomized osteoporotic mice, improve bone microstructure, and regulate calcium and phosphorus metabolism and bone turnover marker levels. Simultaneously, in vitro cell experiments have confirmed that Morkotin A can significantly promote osteoblast differentiation and inhibit osteoclast formation. These in vitro and in vivo experiments have confirmed that Morkotin A has the effect of promoting bone formation and inhibiting bone resorption, and its further development into a drug for the prevention and treatment of osteoporosis, or into a health food or functional food that helps improve bone mineral density, has significant clinical translational value.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The application of Morkotin A in the preparation of pharmaceuticals, health foods or functional foods, the chemical structural formula of Morkotin A is shown in formula (1): Equation (1).

[0006] Preferably, the medicine is a medicine for the prevention and treatment of osteoporosis; the health food or functional food has the health benefits of improving bone density.

[0007] Preferably, Morkotin A can significantly increase bone mineral density in osteoporotic mice; improve the microstructure of trabecular bone in osteoporotic mice, increase bone volume fraction, trabecular bone number and thickness, and reduce trabecular bone separation; regulate serum calcium, phosphorus, and alkaline phosphatase levels in osteoporotic mice, reduce urinary Ca / Cr and P / Cr ratios, thereby reducing bone calcium and phosphorus loss; and significantly reduce the levels of bone turnover markers PINP and CTX-1 in osteoporotic mice, inhibiting their abnormally high bone turnover state.

[0008] Preferably, Morkotin A can enhance osteoblast ALP activity and promote the formation of cellular mineralization nodules.

[0009] Preferably, Morkotin A can inhibit osteoclast TRAP activity and F-actin ring formation.

[0010] A medicine for the prevention and treatment of osteoporosis, including Morkotin A.

[0011] Preferably, the single-dose administration of Morkotin A is 5~30 mg / kg.

[0012] Preferably, the drug dosage form is a pharmaceutically permissible oral dosage form, injectable dosage form, or topical dosage form.

[0013] One type of health supplement that helps improve bone density includes Morkotin A, with a single dose of 5-30 mg / kg.

[0014] A functional food that helps improve bone density includes Morkotin A, with a single dose of 5-30 mg / kg.

[0015] Based on the above technical solutions, compared with existing technologies, the beneficial effects of this invention are as follows: 1) It is the first time that Morkotin A has been shown to have clear anti-osteoporosis activity, promoting bone formation and inhibiting bone resorption, and bidirectionally regulating bone metabolism; 2) In experimental animals, Morkotin A can significantly increase bone density, improve bone microstructure, and regulate calcium and phosphorus metabolism within a dosage range of 5-30 mg / kg; 3) Morkotin A is derived from wolfberry leaves, which are both food and medicine, and has high safety. The above confirms that Morkotin A can be developed into a drug for the prevention and treatment of osteoporosis, or into a health food or functional food that improves bone density, providing a new direction for the high-value utilization of wolfberry leaf resources. Attached Figure Description

[0016] Figure 1 The attached figure shows the effect of Morkotin A on bone mineral density (BMD) of the femur in mice (Sham group, OVX group, Positive group, alendronate sodium group, MA-H group, high-dose Morkotin A group, MA-L group, low-dose Morkotin A group; compared with the sham group, ###p<0.001; compared with the model group, ***p<0.001).

[0017] Figure 2 The attached figure shows the effect of Morkotin A on the three-dimensional reconstruction morphology of mouse femur using Micro-CT (Sham group, OVX group, Positive group, alendronate sodium group, MA-H group, and MA-L group; compared with the sham group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001).

[0018] Figure 3 The attached figure shows the effect of Morkotin A on the microstructural parameters of the femur in mice (Sham is the sham-operated group, OVX is the model group, Positive is the positive control drug alendronate sodium group, MA-H is the high-dose Morkotin A group, and MA-L is the low-dose Morkotin A group; compared with the sham-operated group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001).

[0019] Figure 4 The attached figure shows the effect of Morkotin A on the levels of serum bone turnover markers (PINP, CTX-1, ALP) in mice (Sham group was the sham-operated group, OVX group was the model group, Positive group was the alendronate sodium positive control group, MA-H group was the high-dose Morkotin A group, and MA-L group was the low-dose Morkotin A group; compared with the sham-operated group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001).

[0020] Figure 5The attached figure shows the effect of Morkotin A on the Ca / Cr and P / Cr ratios in mice (Sham is the sham-operated group, OVX is the model group, Positive is the alendronate sodium positive control group, MA-H is the high-dose Morkotin A group, and MA-L is the low-dose Morkotin A group; compared with the sham-operated group, ###p<0.001; compared with the model group, *p<0.05, **p<0.01, ***p<0.001).

[0021] Figure 6 The attached figure shows the effect of Morkotin A on ALP activity in MC3T3-E1 osteoblasts (0 μM is the blank control group, and 0.1, 0.5, and 1 μM are different concentrations of Morkotin A administered; compared with the blank control group, ***p<0.001).

[0022] Figure 7 The attached figure shows the effect of Morkotin A on the formation of mineralized nodules in MC3T3-E1 osteoblasts (0 μM is the blank control group, and 0.1, 0.5, and 1 μM are different concentrations of Morkotin A administered; compared with the blank control group, **p<0.01, ****p<0.0001).

[0023] Figure 8 The attached figure shows the effects of Morkotin A on TRAP activity and F-actin ring formation in RANKL-induced RAW264.7 osteoclasts (Control group was the blank control group, RANKL was the model group, and 1, 0.5, and 0.1 μM were different concentrations of Morkotin A administered; compared with the RANKL model group, **p<0.01, ***p<0.001, ****p<0.0001). Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Morkotin A used in the following examples is the compound shown in formula (1) above, which can be obtained experimentally.

[0026] All reagents used in this invention are commercially available analytical grade; experimental methods not mentioned are conventional experimental methods and will not be described in detail here. Example 1

[0027] A medicine for the prevention and treatment of osteoporosis, in the form of an oral dosage form, comprising Morkotin A, wherein a single dose of Morkotin A is 10 mg / kg. Example 2

[0028] A medicine for the prevention and treatment of osteoporosis, in the form of an oral dosage form, comprising Morkotin A, wherein a single dose of Morkotin A is 30 mg / kg. Example 3

[0029] A drug for the prevention and treatment of osteoporosis, in the form of an injectable formulation, comprising Morkotin A, wherein a single dose of Morkotin A is 5 mg / kg. Example 4

[0030] A medicine for the prevention and treatment of osteoporosis, in the form of a topical patch, comprising Morkotin A, wherein a single application dose of Morkotin A is 15 mg / kg. Example 5

[0031] A health supplement that helps improve bone density, in oral form, includes Morkotin A, wherein a single dose of Morkotin A is 10 mg / kg. Example 6

[0032] A functional food that helps improve bone density, in oral form, includes Morkotin A, wherein a single dose of Morkotin A is 5 mg / kg.

[0033] Experiment 1 The following experiments further illustrate the effects of Examples 1 to 6 above: I. Experimental Design Using an ovariectomized (OVX) induced postmenopausal osteoporosis model in mice, Morkotin A was administered by gavage. The preventive and therapeutic effects of Morkotin A on osteoporosis mice were systematically evaluated using in vitro indicators such as femoral bone mineral density, bone microstructure parameters (BMD, BV / TV, Tb.N, Tb.Th, Tb.Sp), bone metabolism markers (serum ALP, PINP, CTX-1, and urinary Ca / Cr, urinary P / Cr), osteoblast proliferation and differentiation, and osteoclast activation.

[0034] II. Experimental Procedure 1. Animal experiments 1.1 Animal model preparation and grouping for drug administration Forty SPF-grade female C57BL / 6 mice, 6-8 weeks old, were divided into groups of 8 and acclimatized for 3 days at an ambient temperature of 25°C and humidity of 50%-60%. Osteoporosis was induced in mice through bilateral ovariectomy; the sham group underwent only fat removal. Mice were randomly assigned to the sham group (Sham), model group (OVX), alendronate sodium positive control group (Positive), low-dose Morkotin A group (MA-L), and high-dose Morkotin A group (MA-H). One week post-surgery, oral administration of alendronate sodium began. Mice in the sham and model groups were given saline orally. The dose of alendronate sodium was 15 mg / kg / week; the low- and high-dose Morkotin A doses were 5 mg / kg / day and 30 mg / kg / day, respectively, administered via gavage for 12 weeks.

[0035] 1.2 Sample Collection and Testing Twenty-four hours after the last administration, mice in each group were fasted but allowed free access to water for 12 hours. Urine samples were collected after 12 hours in metabolic cages, centrifuged at 3000 r / min for 10 minutes at 4°C, and the supernatant was collected and stored at -80°C for analysis. Blood was collected by enucleation of the mice's eyeballs. After standing at 4°C for 2 hours, the blood was centrifuged at 3000 r / min for 10 minutes, and the supernatant was collected and centrifuged again at 10000 r / min for 5 minutes to separate the serum, which was then stored at -80°C for later use. Mice were euthanized by cervical dislocation after blood collection. The bilateral femurs were quickly dissected, the attached soft tissue was removed, and the femurs were fixed in physiological saline for subsequent bone microstructure analysis.

[0036] 1.3 Bone mineral density and bone microstructure measurement After fixation, femoral samples were collected and scanned using a Micro-CT system, performing continuous tomographic scans of the distal femoral metaphysis. The scan data were then used by the system's built-in software for three-dimensional reconstruction, and quantitative analysis was performed on bone microstructure parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp).

[0037] 1.4 Detection of bone metabolism markers Serum and urine samples stored at -80℃ were thawed at room temperature and then strictly followed the kit instructions: the levels of bone formation markers alkaline phosphatase (ALP), type I procollagen N-terminal peptide (PINP), and bone resorption marker type I collagen C-terminal peptide (CTX-1) in serum were detected by ELISA; the concentrations of calcium (Ca), phosphorus (P), and creatinine (Cr) in serum and urine were measured by a fully automated biochemical analyzer, and the urine Ca / Cr and urine P / Cr ratios were calculated to correct for differences in urine concentration.

[0038] 2. Cell experiments 2.1 Osteoblast assay (MC3T3-E1) Cell proliferation and toxicity: Logarithmic growth phase MC3T3-E1 cells were seeded at 5×10³ / well in 96-well plates. After adhesion, the cells were treated with 0.1, 1, and 10 μM Morkotin A for 48 hours. The absorbance was measured at 450 nm using the CCK-8 assay to calculate cell viability.

[0039] Osteogenic differentiation and ALP activity: Cells were seeded in 24-well plates and osteogenic induction medium (containing sodium β-glycerophosphate, ascorbic acid, and dexamethasone) was administered simultaneously. After 7 days of induction, ALP activity was qualitatively observed by BCIP / NBT staining and ALP expression level was quantitatively detected by enzyme-linked immunosorbent assay (ELISA).

[0040] Detection of mineralized nodules: After 21 days of continuous induction, the nodules were stained with 0.1% Alizarin Red S (pH 4.2) and the morphology of the mineralized nodules was observed under an inverted microscope; after dissolving in 10% hexadecylpyridine chloride, the absorbance was measured at 562 nm to quantitatively analyze the degree of mineralization.

[0041] 2.2 Osteoclast assay (RAW264.7) Osteoclast induction: RAW264.7 cells were inoculated at a rate of 1×10⁻⁶. 4 / well seeding, using induction medium containing M-CSF (50 ng / mL) and RANKL (100 ng / mL), and simultaneously treated with different concentrations of Morkotin A, changing the medium every 2–3 days, inducing multinucleated cell formation in 5–7 days.

[0042] TRAP staining identification: After fixation with 4% paraformaldehyde, the cells were incubated with TRAP staining solution, and positive multinucleated cells with ≥3 nuclei were counted to assess the osteoclast differentiation level.

[0043] F-actin ring structure observation: After cell fixation and permeabilization, F-actin was labeled with FITC-phalloidin, the nucleus was stained with DAPI, and the morphology and integrity of the characteristic actin ring of osteoclasts were observed under a laser confocal microscope.

[0044] 3. Data Statistics This invention uses Graphpad Prism 9.5 software for data processing and statistical analysis. All results are expressed as mean ± standard deviation (X±S); one-way ANOVA was used for comparisons between groups, with p < 0.05 considered statistically significant.

[0045] III. Experimental Results 1. Effect on bone mineral density of mouse femur The effect of Morkotin A on femoral bone mineral density in mice is shown in the following results. Figure 1As shown in the figure, compared with the sham-operated group (115.23±5.17 mg / cm³), the femoral bone mineral density of mice in the model group (64.89±4.92 mg / cm³) was significantly reduced (p<0.001), indicating that the OVX-induced osteoporosis mouse model was successful. Compared with the model group, the femoral bone mineral density of mice in the positive control group (alendronate sodium, 254.78±12.35 mg / cm³) was significantly increased (p<0.001), and the BMD of the high-dose Morkotin A group (MA-H, 119.75±6.24 mg / cm³) and the low-dose Morkotin A group (MA-L, 109.82±5.86 mg / cm³) was significantly increased (p<0.001), and the increase was dose-dependent.

[0046] In summary, osteoporosis significantly reduces femoral bone mineral density in mice. Different dosage groups of Morkotin A were able to increase femoral bone mineral density in mice to varying degrees.

[0047] 2. Effects of Morkotin A on femoral bone microstructure in OVX mice The effects of Morkotin A on the femoral bone microstructure of each group of mice are shown in the following results. Figure 2 As shown, in the sham-operated group (Sham), the distal femoral trabeculae of mice were densely arranged and well-connected, with a uniform network distribution and smooth, continuous cortical bone. In the model group (OVX), the number of femoral trabeculae was significantly reduced, their arrangement was sparse, and fractures were increased. The intertrabecular gaps were significantly enlarged, and the cortical bone was thinned, indicating severe damage to the bone microstructure in the OVX-induced osteoporosis model. Compared with the model group, the positive control group (Positive) showed a significant increase in trabecular bone density, a more regular arrangement, and significantly improved trabecular bone connectivity. The high-dose Morkotin A group (MA-H) and low-dose Morkotin A group (MA-L) showed a significantly increased number of trabeculae, reduced trabecular bone fractures, and smaller gaps. The improvement effect of the MA-H group was closer to that of the positive control group, indicating that Morkotin A can effectively improve the bone microstructure damage in OVX mice and alleviate the pathological changes of osteoporosis.

[0048] 3. Effects on microstructural parameters of mouse femur The effects of Morkotin A on mouse femoral bone microstructural parameters are shown in the following results. Figure 3 .like Figure 3 As shown in Figure A, compared with the sham surgery group (4.18±0.35%), the bone volume fraction (BV / TV) in the model group (1.52±0.21%) was significantly lower (p<0.001); compared with the model group, the positive control group (21.95±1.87%) was significantly higher (p<0.001), and the MA-H group (3.76±0.42%) and MA-L group (3.48±0.39%) were significantly higher (p<0.01, p<0.05).

[0049] like Figure 3 As shown in B, compared with the sham surgery group (0.89±0.07 1 / mm), the number of trabecular bone (Tb.N) in the model group (0.31±0.04 1 / mm) was significantly decreased (p<0.001); compared with the model group, the positive drug group (3.08±0.25 1 / mm) was significantly increased (p<0.001), and the MA-H group (0.78±0.06 1 / mm) and MA-L group (0.69±0.05 1 / mm) were significantly increased (p<0.01, p<0.05).

[0050] like Figure 3 As shown in Figure C, compared with the sham surgery group (309.24±15.62μm), the trabecular separation (Tb.Sp) in the model group (478.56±18.73μm) was significantly increased (p<0.001); compared with the model group, the positive control group (198.65±12.47μm) was significantly decreased (p<0.001), and the MA-H group (328.72±16.35μm) and MA-L group (348.17±17.21μm) were significantly decreased (p<0.01, p<0.05).

[0051] like Figure 3 As shown in Figure D, compared with the sham surgery group (41.87±2.13μm), the trabecular bone thickness (Tb.Th) in the model group (30.12±2.05μm) was significantly decreased (p<0.001); compared with the model group, the positive control group (67.83±3.12 μm) was significantly increased (p<0.001), and the MA-H group (43.91±2.56μm) and MA-L group (39.85±2.34μm) were significantly increased (p<0.01, p<0.05).

[0052] 4. Effects on serum bone turnover marker levels in mice The effect of Morkotin A on serum bone turnover markers in mice is shown in the following results. Figure 4 .like Figure 4 As shown in Figure A, compared with the sham-operated group (1.65±0.08 mmol / L), the serum calcium level in the model group mice was slightly decreased (1.62±0.10 mmol / L), but the difference was not statistically significant. Compared with the model group, the serum calcium levels in the positive control group (1.63±0.09 mmol / L), MA-H group (1.61±0.07 mmol / L), and MA-L group (1.58±0.08 mmol / L) showed no significant changes, indicating that Morkotin A has no significant effect on serum calcium homeostasis.

[0053] like Figure 4As shown in Figure B, compared with the sham-operated group (2.30±0.15 mmol / L), the serum P level in the model group mice was slightly increased (2.40±0.20 mmol / L), but the difference was not statistically significant. Compared with the model group, the serum P level in the positive control group (2.05±0.12 mmol / L) was significantly decreased (p<0.05), while the serum P levels in the MA-H group (2.50±0.18 mmol / L) and MA-L group (2.35±0.22 mmol / L) showed no significant change.

[0054] like Figure 4 As shown in Figure C, compared with the sham-operated group (75.00±10.00 U / L), the serum ALP level in the model group mice was significantly increased (95.00±10.00 U / L, p<0.05), indicating abnormally enhanced bone formation activity. Compared with the model group, the serum ALP levels in the positive control group (80.00±10.00 U / L, p<0.05), MA-H group (85.00±10.00 U / L, p<0.05), and MA-L group (78.00±10.00 U / L, p<0.05) were significantly decreased, showing a dose-dependent recovery trend.

[0055] like Figure 4 As shown in Figure D, compared with the sham-operated group (200.00±50.00 pg / mL), the serum CTX-1 level in the model group mice was significantly increased (500.00±70.00 pg / mL, p<0.001), indicating a significant enhancement in bone resorption activity. Compared with the model group, the serum CTX-1 levels in the positive control group (220.00±40.00 pg / mL, p<0.001), MA-H group (280.00±50.00 pg / mL, p<0.01), and MA-L group (380.00±60.00 pg / mL, p<0.05) were significantly decreased, showing a dose-dependent inhibition of bone resorption.

[0056] like Figure 4 As shown in Figure E, compared with the sham-operated group (2600.00±300.00 pg / mL), the serum PINP level in the model group mice was significantly increased (4800.00±400.00 pg / mL, p<0.001), indicating hyperactive bone formation activity. Compared with the model group, the serum PINP levels in the positive control group (2900.00±300.00 pg / mL, p<0.001), MA-H group (3400.00±350.00 pg / mL, p<0.01), and MA-L group (3900.00±400.00 pg / mL, p<0.05) were significantly decreased, showing a dose-dependent inhibition of abnormal bone formation.

[0057] In summary, the levels of bone turnover markers (ALP, CTX-1, PINP) in the serum of OVX model mice were significantly elevated, indicating a state of high bone turnover. Morkotin A at different doses could restore the levels of bone turnover markers to varying degrees, effectively inhibiting abnormal bone turnover and balancing bone formation and bone resorption.

[0058] 5. Effects on calcium and phosphorus metabolism indicators in mouse serum and urine The effects of Morkotin A on calcium and phosphorus metabolism parameters in mouse serum and urine are shown in the following results. Figure 5 .like Figure 5 As shown in Figure A, compared with the sham-operated group (0.0006±0.0001), the urinary Ca / Cr ratio in the model group mice (0.0010±0.0002) was significantly increased (p<0.01), indicating increased bone calcium loss in the model mice. Compared with the model group, the urinary Ca / Cr ratio in the positive control group (0.0006±0.0001) was significantly decreased (p<0.01); the urinary Ca / Cr ratios in both the MA-H group (0.0007±0.0001) and the MA-L group (0.0007±0.0001) were significantly decreased (p<0.05), suggesting that Morkotin A can reduce urinary calcium excretion and decrease bone calcium loss in osteoporotic mice.

[0059] like Figure 5 As shown in Figure B, compared with the sham-operated group (0.013±0.002), the urinary P / Cr ratio in the model group mice (0.022±0.003) was significantly increased (p<0.01), indicating increased bone phosphorus loss in the model mice. Compared with the model group, the urinary P / Cr ratio in the positive control group (0.014±0.002) was significantly decreased (p<0.05); the urinary P / Cr ratio in the high-dose Morkotin A group (0.018±0.002) was significantly decreased (p<0.05), while the low-dose group (0.021±0.003) showed a decreasing trend but the difference was not statistically significant, suggesting that Morkotin A can reduce bone phosphorus loss to a certain extent and improve calcium and phosphorus metabolism disorders.

[0060] In summary, osteoporosis model mice exhibit a significant imbalance in calcium and phosphorus metabolism. Different doses of Morkotin A improved calcium and phosphorus metabolism and reduced bone calcium loss to varying degrees.

[0061] No mice died during the entire administration process, and no abnormalities were observed in their appearance or behavior.

[0062] 6. Effects on ALP activity in MC3T3-E1 osteoblasts The effect of Morkotin A on ALP activity in MC3T3-E1 osteoblasts is shown in the following results. Figure 6As shown in the figure, compared with the normal control group (0 μM, 0.23±0.04 mM), the ALP activities in the 0.1 μM, 0.5 μM, and 1 μM Morkotin A treatment groups were 0.45±0.03 mM, 0.46±0.03 mM, and 0.47±0.04 mM, respectively, all significantly increased (p<0.001), and showed an increasing trend with increasing drug concentration, suggesting that Morkotin A can significantly promote early osteoblast differentiation. ALP staining results showed that only a small amount of blue-purple positive staining was observed in the control group cells; as the Morkotin A concentration increased, the blue-purple staining area gradually increased and the color deepened, and the number of positive cells increased significantly, consistent with the quantitative results, indicating the promoting effect of Morkotin A on osteoblast differentiation.

[0063] 7. Effects on the formation of mineralized nodules in MC3T3-E1 osteoblasts The effect of Morkotin A on the formation of mineralized nodules in MC3T3-E1 osteoblasts is shown in the following results. Figure 7 Compared with the control group (0 μM, mineralization: 0.22±0.04), the mineralization of the 0.1 μM, 0.5 μM, and 1 μM Morkotin A treatment groups were 0.41±0.05, 0.41±0.04, and 0.55±0.06, respectively, all significantly increased (p<0.01, p<0.0001), and increased in a concentration-dependent manner. Alizarin Red S staining results showed that only a small number of red mineralized nodules were observed in the control group; with increasing drug concentration, the number, size, and color of the red nodules increased, consistent with the quantitative results, indicating the promoting effect of Morkotin A on osteoblast mineralization and maturation.

[0064] In summary, Morkotin A can significantly promote the formation of mineralized nodules in MC3T3-E1 cells and accelerate the osteoblast maturation process.

[0065] 8. Effects on TRAP activity and F-actin loop formation in RAW264.7 osteoclasts The effects of Morkotin A on TRAP activity and F-actin ring formation in RAW264.7 osteoclasts are shown in the following results. Figure 8 As shown. Compared with the normal control group, the model group showed significantly increased TRAP activity and the number of TRAP-positive multinucleated cells in osteoclasts (p<0.001), and a significant increase in F-actin ring formation. Compared with the model group, the positive drug group showed significantly decreased TRAP activity and the number of TRAP-positive multinucleated cells (p<0.001); all Morkotin A dose groups showed significantly decreased TRAP activity and the number of TRAP-positive multinucleated cells (p<0.05, p<0.001), and a significant reduction in F-actin ring formation, showing a concentration-dependent inhibitory trend.

[0066] In summary, Morkotin A at different dosages inhibited osteoclast differentiation and bone resorption activity to varying degrees.

[0067] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0068] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of morkotin A for the preparation of a pharmaceutical product, a health food product or a functional food product, characterized in that, The Morkotin A, also known as quercetin-3-O-rutin-7-O-glucoside, has the structure shown in formula (1): Formula (1).

2. Use according to claim 1, wherein The medicine is for the prevention and treatment of osteoporosis; the health food or functional food has the health benefits of improving bone density.

3. The use according to claim 1, wherein Morkotin A can increase bone mineral density and improve the trabecular microstructure of osteoporotic mice.

4. The use according to claim 1, wherein Morkotin A can regulate serum calcium, phosphorus, and bone turnover marker levels in osteoporotic mice.

5. The use according to claim 1, wherein Morkotin A can promote osteoblast proliferation and mineralization.

6. The application as described in claim 1, characterized in that, Morkotin A can inhibit osteoclast differentiation and bone resorption activity.

7. A medicine for the prevention and treatment of osteoporosis, characterized in that, Including Morkotin A, the single-dose administration is 5~30mg / kg.

8. The pharmaceutical product as described in claim 7, characterized in that, The dosage form can be oral, injectable, or topical.

9. A health food product that helps improve bone density, characterized in that, Including Morkotin A, the single-dose administration is 5~30mg / kg.

10. A functional food that helps improve bone density, characterized in that, Including Morkotin A, the single-dose administration is 5~30mg / kg.

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