Application of total dandelion flavonoids in the preparation of products for the prevention and treatment of osteoporosis

Abstract

This invention relates to the field of dandelion total flavonoids application technology, and discloses the application of dandelion total flavonoids in the preparation of products for the prevention and treatment of osteoporosis. The extraction method of dandelion total flavonoids according to this invention includes the following steps: 1) drying and pulverizing the whole dandelion plant to obtain dandelion powder; 2) mixing the dandelion powder with 60% ethanol (volume fraction), ultrasonically treating, and then filtering; 3) mixing the filtered residue with 40% ethanol (volume fraction), heating and refluxing, and then filtering; 4) combining the filtrates, concentrating, and drying to obtain dandelion total flavonoids. This invention, through biological and cellular level studies, has found that dandelion total flavonoids can effectively improve bone density and bone quality; therefore, dandelion total flavonoids can be applied to products for the prevention and treatment of osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of dandelion total flavonoids application technology, and more specifically, to the application of dandelion total flavonoids in the preparation of products for the prevention and treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a systemic bone disease caused by decreased bone density and quality, destruction of bone microstructure, and increased bone fragility, making it prone to fractures. Symptoms of osteoporosis include pain, spinal deformities, and fragility fractures, which pose serious risks to the body. Pain itself can reduce the patient's quality of life, spinal deformities and fractures can lead to disability, and increase the incidence of lung infections and bedsores, thus increasing the patient's quality of life and mortality.

[0003] Dandelion, a perennial herb belonging to the Asteraceae family and the Taraxacum genus, contains various components such as taraxerol, taraxacin, choline, and flavonoids. It has long been mainly used in products for the prevention and treatment of diseases such as heat toxicity, pain, and inflammation, and no research has found that it can be used in products for the prevention and treatment of osteoporosis. Summary of the Invention

[0004] The purpose of this invention is to provide an application of total dandelion flavonoids in the preparation of products for the prevention and treatment of osteoporosis. Total dandelion flavonoids can improve bone density and bone quality, thus effectively being used in products for the prevention and treatment of osteoporosis.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Application of total flavonoids from dandelion in the preparation of products for the prevention and treatment of osteoporosis.

[0007] Furthermore, the extraction method for total flavonoids from dandelion includes the following steps:

[0008] 1) Dry the whole dandelion plant, pulverize it, and obtain dandelion powder;

[0009] 2) Mix the dandelion powder obtained in step 1) with 60% ethanol by volume, sonicate, and then filter.

[0010] 3) Mix the filter residue from step 2) with 40% ethanol by volume, heat under reflux, and then filter.

[0011] 4) Combine the filtrates obtained in steps 2) and 3), concentrate and dry them to obtain the total dandelion flavonoids.

[0012] Furthermore, in step 1), the drying process involves drying until the moisture content is less than 20%.

[0013] Furthermore, in step 1), the pulverization is pulverizing to pass through a 40-mesh sieve.

[0014] Furthermore, in step 2), the ratio of dandelion powder to ethanol with a volume fraction of 60% is 1:(25-35).

[0015] Furthermore, in step 2), the ultrasonic treatment power is 120W, the temperature is 70-90℃, and the time is 25-35min.

[0016] Furthermore, in step 3), the ratio of the filter residue to ethanol with a volume fraction of 40% is 1:(15-25).

[0017] Furthermore, in step 3), the temperature of the heating reflux treatment is 55-65°C, and the time is 1-3 hours.

[0018] Furthermore, in step 4), the drying process involves drying until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] This invention, through research at the biological and cellular levels, has found that total flavonoids from dandelion can effectively improve bone density and bone quality. Therefore, total flavonoids from dandelion can be used in products for the prevention and treatment of osteoporosis. Attached Figure Description

[0021] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention.

[0022] Figure 1 The results of the average weight gain of mice are shown in Figure A, where the initial average weight of mice in each group is shown in Figure B, and the average weight gain of mice in each group after the drug administration is completed.

[0023] Figure 2 Image showing the results of quantitative analysis of bone mineral density in mouse femur;

[0024] Figure 3 The results of quantitative analysis of bone volume fraction are shown in the figure.

[0025] Figure 4 Image showing the results of quantitative analysis of bone surface area density;

[0026] Figure 5 This is a microstructure diagram of the mouse femur after three-dimensional reconstruction.

[0027] Figure 6 Figure showing the results of quantitative analysis of trabecular pattern factors;

[0028] Figure 7 This is a graph showing the results of quantitative porosity analysis.

[0029] Figure 8 A graph showing the quantitative analysis results of the number of trabeculae;

[0030] Figure 9 The graph shows the quantitative analysis results of the beam gap width.

[0031] Figure 10 ABH / OG staining pattern;

[0032] Figure 11 Figure showing the results of quantitative analysis of ABH / OG stained trabecular bone area;

[0033] Figure 12 HE staining diagram;

[0034] Figure 13 Image showing the results of quantitative analysis of bone trabecular area stained with HE;

[0035] Figure 14 A TRAP staining diagram;

[0036] Figure 15 Figure showing the results of quantitative analysis of TRAP-positive cells around the distal femoral growth plate;

[0037] Figure 16 ALP staining diagram;

[0038] Figure 17 Image showing the results of quantitative analysis of the area of ​​ALP-positive staining around the distal femoral growth plate;

[0039] Figure 18 This is a graph showing the results of a urinary calcium test.

[0040] Figure 19 The image shows the results of magnesium testing in urine.

[0041] Figure 20 The image shows the results of phosphorus detection in urine.

[0042] Figure 21 Figure 1 shows the effect of different concentrations of total dandelion flavonoids on the cell viability of RAW264.7 cells.

[0043] Figure 22 Figure showing the effect of different culture medium conditions on the cell viability of MC3T3-E1 cells;

[0044] Figure 23 This is a graph showing the expression levels of osteogenic differentiation-related genes BGLAP, SP7, ALP, COL1, and Bsp.

[0045] Figure 24This is a representative band diagram showing the expression of osteogenic differentiation-related proteins BGLAP, SP7, ALP, and COL1;

[0046] Figure 25 This is a graph showing the protein expression levels of BGLAP, SP7, ALP, and COL1.

[0047] Figure 26 This is a graph showing the expression level of Col16a1, a gene related to the bone mineralization pathway.

[0048] Figure 27 This is a graph showing the expression level of Isg15, a gene associated with the bone mineralization pathway.

[0049] Figure 28 This is a graph showing the expression levels of Clec3b, a gene associated with the bone mineralization pathway.

[0050] Figure 29 This is a graph showing the expression levels of Ptn, a gene associated with the bone mineralization pathway.

[0051] Figure 30 This is a graph showing the expression level of Matn1, a gene associated with the bone mineralization pathway. Detailed Implementation

[0052] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0053] In the following embodiments, the extraction method of total flavonoids from dandelion includes the following steps:

[0054] 1) Dry the whole dandelion plant until the moisture content is less than 20%, crush it, and pass it through a 40-mesh sieve to obtain dandelion powder;

[0055] 2) Mix the dandelion powder obtained in step 1) with 60% ethanol at a material-to-liquid ratio of 1:(25-35), sonicate, and then filter.

[0056] The ultrasonic treatment involved a power of 120W, a temperature of 70–90℃, and a duration of 25–35 minutes.

[0057] 3) Mix the filter residue from step 2) with 40% ethanol at a ratio of 1:(15-25), heat and reflux, and then filter.

[0058] The reflux heating process is carried out at a temperature of 55–65°C for 1–3 hours.

[0059] 4) Combine the filtrates obtained in steps 2) and 3), concentrate them, and dry them until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%, to obtain the total flavonoids of dandelion.

[0060] Example 1

[0061] Extraction of total flavonoids from dandelion

[0062] 1) Dry the whole dandelion plant until the moisture content is less than 20%, crush it, and pass it through a 40-mesh sieve to obtain dandelion powder;

[0063] 2) Mix the dandelion powder obtained in step 1) with 60% ethanol at a ratio of 1:30, sonicate, and then filter.

[0064] The ultrasonic treatment involved a power of 120W, a temperature of 80℃, and a duration of 30 minutes.

[0065] 3) Mix the filter residue from step 2) with 40% ethanol at a ratio of 1:20, heat and reflux, and then filter.

[0066] The reflux heating process was carried out at a temperature of 60°C for 2 hours.

[0067] 4) Combine the filtrates obtained in steps 2) and 3), concentrate them, and dry them until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%, to obtain the total flavonoids of dandelion.

[0068] The total flavonoid extraction rate of dandelion in Example 1 was calculated to be 37.81%.

[0069] Example 2

[0070] Extraction of total flavonoids from dandelion

[0071] 1) Dry the whole dandelion plant until the moisture content is less than 20%, crush it, and pass it through a 40-mesh sieve to obtain dandelion powder;

[0072] 2) Mix the dandelion powder obtained in step 1) with 60% ethanol at a material-to-liquid ratio of 1:25, sonicate, and then filter.

[0073] The ultrasonic treatment involved a power of 120W, a temperature of 70℃, and a duration of 25 minutes.

[0074] 3) Mix the filter residue from step 2) with 40% ethanol at a ratio of 1:15, heat and reflux, and then filter.

[0075] The reflux heating process was carried out at a temperature of 55°C for 1 hour.

[0076] 4) Combine the filtrates obtained in steps 2) and 3), concentrate them, and dry them until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%, to obtain the total flavonoids of dandelion.

[0077] The total flavonoid extraction rate of dandelion in Example 2 was calculated to be 33.54%.

[0078] Example 3

[0079] Extraction of total flavonoids from dandelion

[0080] 1) Dry the whole dandelion plant until the moisture content is less than 20%, crush it, and pass it through a 40-mesh sieve to obtain dandelion powder;

[0081] 2) Mix the dandelion powder obtained in step 1) with 60% ethanol at a ratio of 1:35, sonicate, and then filter.

[0082] The ultrasonic treatment involved a power of 120W, a temperature of 90℃, and a duration of 35 minutes.

[0083] 3) Mix the filter residue from step 2) with 40% ethanol at a ratio of 1:25, heat and reflux, and then filter.

[0084] The reflux heating process was carried out at a temperature of 65°C for 3 hours.

[0085] 4) Combine the filtrates obtained in steps 2) and 3), concentrate them, and dry them until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%, to obtain the total flavonoids of dandelion.

[0086] The total flavonoid extraction rate of dandelion in Example 3 was calculated to be 35.65%.

[0087] Effect Verification: The Effects of Total Flavonoids from Dandelion on the Body's Bones

[0088] (I) Selection and Adaptation of Test Mice

[0089] Fifty healthy 9-week-old C57BL / 6 mice with similar body weight (22.61±2.28g) were randomly selected and randomly divided into four groups: filtered air exposure + distilled water group (FC), filtered air exposure + high dose of dandelion total flavonoids group (FH), PM2.5 concentrated air exposure + distilled water group (PC), PM2.5 concentrated air exposure + low dose of dandelion total flavonoids group (PL), and PM2.5 concentrated air exposure + high dose of dandelion total flavonoids group (PH). The mice were then acclimatized in a clean culture chamber for 7 days, during which they had free access to food and water.

[0090] The air is filtered as follows: air that has been filtered through a HEPA high-efficiency filter to remove more than 95% of the particulate matter in the air;

[0091] PM2.5 condensed air is air whose PM2.5 concentration is 3 to 6 times that of the ambient PM2.5 concentration through a particulate matter concentration system.

[0092] (II) Selection of test substance and administration method

[0093] The total dandelion flavonoids extracted in Example 1 were dissolved in 0.3 mL of distilled water and administered by gavage according to the prescribed dosage.

[0094] (III) Drug administration

[0095] 1) Pre-treatment: Mice in each group were housed under filtered air and allowed free access to food and water. During this period, mice in the PL group were given 50 mg / kg of total dandelion flavonoids by gavage daily, mice in the FH and PH groups were given 150 mg / kg of total dandelion flavonoids by gavage daily, and mice in the FC and PC groups were given an equal volume of distilled water by gavage daily for 7 days.

[0096] 2) Administration: Mice in the FC and FH groups were placed in a clean culture chamber with filtered air introduced, while mice in the PC, PL, and PH groups were placed in a PM2.5 concentrated exposure culture chamber with PM2.5 concentrated air introduced. The environmental conditions of the two chambers were kept consistent, including temperature (22±2℃), air pressure (100kPa), and light (12h light / 12h darkness). Mice in the PL group were given 50mg / kg of total dandelion flavonoids by gavage daily, mice in the FH and PH groups were given 150mg / kg of total dandelion flavonoids by gavage daily, and mice in the FC and PC groups were given an equal volume of distilled water by gavage daily for six weeks.

[0097] (iv) Sample preparation

[0098] After the administration of the drug, the mice in each group were euthanized, the weight of each group of mice was recorded, the urine of the mice was collected and stored at -80℃ and sent to the laboratory for later use, and the right femur of each group of mice was collected and sent to the laboratory for later use.

[0099] (V) Weight gain measurement

[0100] Calculate the average weight gain of mice in each group; the results of the average weight gain measurement are as follows: Figure 1 As shown in the figure, Figure A shows the initial average weight of mice in each group, and Figure B shows the average weight gain of mice in each group after the drug administration.

[0101] Depend on Figure 1It can be seen that there was no significant statistical difference in the average weight gain of mice in each group. Therefore, it can be concluded that the total flavonoids extracted from dandelion in this invention will not have a negative impact on the growth performance of the body when used in the preparation of products for the prevention and treatment of osteoporosis.

[0102] (vi) Bone mineral density measurement

[0103] The right femur of mice was fixed with 4% paraformaldehyde and then subjected to Micro-CT scanning. Quantitative analysis of femoral bone mineral density was performed on each group of mice. The results of the quantitative analysis of femoral bone mineral density are as follows: Figure 2 As shown in the figure, * indicates P < 0.05;

[0104] Bone mineral density (BMD) refers to the density of bone minerals per unit volume of bone. It reflects the strength of bones and is a commonly used indicator for assessing bone health. It can be used to diagnose osteoporosis and assess fracture risk. When BMD is below the normal range, it indicates the presence of osteoporosis or other bone health problems.

[0105] Depend on Figure 2 The results showed that the femoral bone mineral density (BMD) of the PC group mice was significantly lower than that of the FC group mice, indicating that PM2.5 exposure caused a decrease in bone mineral density in mice. In addition, the femoral bone BMD of the PC group mice was significantly lower than that of the PL and PH groups mice, indicating that administration of total dandelion flavonoids could effectively increase bone mineral density in mice and significantly improve the problem of decreased bone mineral density caused by PM2.5 exposure.

[0106] (vii) Bone mass measurement

[0107] The right femur of mice was fixed with 4% paraformaldehyde and then subjected to Micro-CT scanning. The femoral bone mass of each group of mice was quantitatively analyzed. The results of the quantitative analysis of femoral bone mass are as follows: Figures 3-4 As shown, where, Figure 3 This is a graph showing the results of quantitative analysis of bone volume fraction. Figure 4 The graph shows the results of quantitative analysis of bone surface area density. In the graph, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0108] Bone volume fraction (BV / TV) describes the proportion of the actual bone mass in bone tissue to the total volume. It is used to assess the density and quality of bone tissue and is an important parameter for assessing bone health. The higher the BV / TV, the greater the proportion of actual bone mass in bone tissue, and the higher the bone density and bone mass.

[0109] Bone surface area density (BS / TV) refers to the surface area of ​​bone tissue per unit volume. It is used to assess the microstructure of bone tissue and the distribution of bone mass. It is an important parameter for assessing bone health. The higher the BS / TV, the larger the bone surface area per unit volume, reflecting an increase in bone mass or a change in the microstructure of bone tissue.

[0110] Depend on Figures 3-4 The results showed that the BV / TV and BS / TV of the PC group mice were significantly lower than those of the FC group mice, indicating that PM2.5 exposure would reduce femoral bone mass in mice. In addition, the BV / TV and BS / TV of the PC group mice were significantly lower than those of the PL and PH groups, indicating that administration of total dandelion flavonoids could effectively increase femoral bone mass in mice and significantly improve the problem of reduced femoral bone mass caused by PM2.5 exposure.

[0111] (viii) Bone microstructure detection

[0112] The right femur of mice was fixed with 4% paraformaldehyde and then subjected to Micro-CT scanning. The bone microstructure of each group of mice was analyzed; the results of the bone microstructure analysis are as follows: Figures 5-9 As shown, where, Figure 5 This is a microstructure diagram of the mouse femur after three-dimensional reconstruction. Figure 6 The figure shows the results of quantitative analysis of trabecular pattern factors. Figure 7 This is a graph showing the results of quantitative porosity analysis. Figure 8 The graph shows the results of the quantitative analysis of the number of trabeculae. Figure 9 The graph shows the quantitative analysis results of the beam gap width. In the graph, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0113] Trabecular Pattern Factor (Tb.PF) is a parameter that describes the structure of the trabecular bone network. It is used to assess the connectivity and arrangement of trabeculae. Understanding Tb.PF can help us understand the microstructure of bone tissue and the health status of bones. The lower the Tb.PF value, the more connected and ordered the trabeculae are. The higher the Tb.PF value, the more gaps and irregularities exist between the trabeculae.

[0114] Porosity describes the degree of voids or pores in bone tissue, which affects the strength and quality of bone.

[0115] Trabecular number (Tb.N) is a parameter describing the density of trabecular bone structure. It is used to assess the number and arrangement density of trabeculae. By understanding Tb.N, we can understand the microstructure of bone tissue and the health status of bones. The higher the Tb.N value, the greater the number density of trabeculae, which is positively correlated with bone mineral density and bone quality.

[0116] Trabecular spacing (Tb.Sp) is a parameter describing the distance between trabeculae and is used to assess the spacing between trabeculae. By understanding Tb.Sp, we can understand the microstructure of bone tissue and the health status of bones. The larger the Tb.Sp value, the wider the spacing between trabeculae and the more sparse the trabeculae are; the smaller the Tb.Sp value, the more compact the trabeculae are.

[0117] Depend on Figures 5-9 It can be seen that the Tb.N of the PC group mice was significantly lower than that of the FC group, while the Tb.N of the PL and PH groups mice was significantly higher than that of the PC group; the Pros ity and Tb.Pf of the PC group mice were significantly higher than those of the FC group, while the Pros ity and Tb.Pf of the PL and PH groups mice were significantly lower than those of the PC group; the Tb.Sp of the PC group mice showed an increasing trend compared with the FC group, while the Tb.Sp of the PL group mice was significantly lower than that of the PC group.

[0118] To further analyze the effects of total dandelion flavonoids on the femoral structure of mice, pathological sections of the mouse femur were stained with ABH / OG and HE, and the area of ​​trabecular bone around the femoral growth plate was quantitatively analyzed. The staining results and the quantitative analysis results of the trabecular bone area are as follows: Figures 10-13 As shown, where, Figure 10 ABH / OG staining pattern Figure 11 This is a graph showing the results of quantitative analysis of ABH / OG stained trabecular bone area. Figure 12 For HE staining diagram, Figure 13 The figure shows the results of quantitative analysis of bone trabecular area stained with HE. * indicates P<0.05, and *** indicates P<0.001.

[0119] Depend on Figures 10-13 It can be seen that both staining methods showed that the trabecular bone area in the PC group was significantly lower than that in the FC group, and the trabecular bone area in the PL and PH groups was significantly increased compared with that in the PC group.

[0120] Therefore, bone microstructure analysis showed that PM2.5 exposure caused abnormalities in the femoral bone microstructure of mice, and administration of total flavonoids from dandelion could significantly improve the microstructural abnormalities caused by PM2.5 exposure in mice.

[0121] (ix) Bone resorption measurement

[0122] TRAP staining was performed around the growth plate of the mouse femur, and TRAP-positive cells around the distal growth plate of the mouse femur were quantitatively analyzed. The TRAP staining results are as follows: Figure 14 As shown, the quantitative analysis results of TRAP-positive cells around the distal femoral growth plate in mice are as follows: Figure 15 As shown in the figure, * indicates P < 0.05;

[0123] Depend on Figures 14-15 It can be seen that the TRAP staining positive area in the PC group was significantly higher than that in the FC group, indicating that PM2.5 exposure increased the number of osteoclasts in the mouse femur and increased the level of bone resorption. However, there was no statistically significant difference between the FC group and the FH group under filtered air conditions. In addition, the TRAP staining positive area in the low-dose and high-dose administration groups (PL group and PH group) under PM2.5 concentrated air conditions was significantly lower than that in the PC group, indicating that the number of osteoclasts in the PL group and PH group was reduced and the level of bone resorption was decreased.

[0124] Therefore, bone resorption analysis showed that PM2.5 exposure caused an increase in bone resorption levels in mice, while administration of total flavonoids from dandelion could significantly improve the increased bone resorption levels caused by PM2.5 exposure.

[0125] (x) Bone formation assay

[0126] ALP staining was performed around the growth plate of the mouse femur, and the area of ​​ALP-positive staining around the distal growth plate of the mouse femur was quantitatively analyzed. The ALP staining results are as follows: Figure 16 As shown, the quantitative analysis results of the ALP-positive area around the distal femoral growth plate in mice are as follows: Figure 17 As shown in the figure, * indicates P < 0.05, and ** indicates P < 0.01;

[0127] Depend on Figures 16-17 It can be seen that the ALP staining positive area in the PC group was significantly lower than that in the FC group; while there was no statistically significant difference between the FC group and the FH group under filtered air conditions; in addition, the ALP staining positive area in the low-dose TF administration groups (PL group and PH group) under PM2.5 concentrated air conditions was significantly higher than that in the PC group.

[0128] Therefore, bone formation analysis showed that the level of femoral bone formation in mice decreased after PM2.5 exposure, and administration of total flavonoids from dandelion could significantly improve the decrease in bone formation caused by PM2.5 exposure.

[0129] (xi) Determination of mineral loss in mouse urine

[0130] Biochemical indicators were tested in the urine of mice transferred to the laboratory;

[0131] The method for detecting calcium in urine is as follows:

[0132] 1) Sample preparation: Dilute the urine sample 10 times with ddH2O;

[0133] 2) Preparation of Ca colorimetric working solution: Before use, mix equal volumes of Ca Assay Buffer and OCPC colorimetric solution from the calcium detection kit to obtain the Ca colorimetric working solution. It should be prepared and used immediately and stored at 4°C in the dark.

[0134] 3) Sample addition: Use a 96-well plate that has been treated with dilute hydrochloric acid and cleaned with deionized water. Set up blank wells, standard wells, and test wells according to Table 1, and add the solution in sequence.

[0135] Table 1. Urine calcium detection

[0136]

[0137] 4) Ca determination: Mix well, let stand at room temperature for 10 min, zero the instrument with the blank well, measure the absorbance of the standard tube and the test tube at 575 nm using an ELISA reader, and calculate the calcium concentration in the urine.

[0138] The calculation formula is: Calcium (mmol / L) = (A 测定 / A 标准 )×2.5×10;

[0139] The results of the urine calcium test are as follows: Figure 18 As shown in the figure, * indicates P < 0.05;

[0140] The method for detecting magnesium in urine is as follows:

[0141] 1) Sample preparation: Dilute the urine sample 10 times with ddH2O;

[0142] 2) Preparation of Mg colorimetric working solution: Before use, mix equal volumes of Mg Assay Buffer and MTB colorimetric solution from the magnesium detection kit to obtain the Mg colorimetric working solution. It should be prepared and used immediately and stored at 4°C in the dark.

[0143] 3) Sample addition: Use a 96-well plate that has been treated with dilute hydrochloric acid and cleaned with deionized water. Set up blank wells, standard wells, and test wells according to Table 2, and add the solution in sequence.

[0144] Table 2. Urinary magnesium detection

[0145]

[0146] 4) Mg determination: Mix well, let stand at room temperature for 5 min, zero the instrument with the blank well, measure the absorbance of the standard tube and the test tube at 600 nm using an ELISA reader, and calculate the magnesium concentration in the urine.

[0147] The calculation formula is: Magnesium (mmol / L) = (A 测定 / A 标准 )×0.823×10;

[0148] The results of the urine magnesium test are as follows: Figure 19 As shown;

[0149] The method for detecting phosphorus in urine is as follows:

[0150] 1) Sample preparation: Dilute the urine sample 10 times with ddH2O, take 0.02 mL of the diluted urine sample, add 0.48 mL of Pi Assay buffer; mix thoroughly and let stand at room temperature for 10 min, then centrifuge at 3000 g for 10 min, and collect the supernatant for subsequent detection.

[0151] 2) Preparation of phosphorus standard working solution: Take an appropriate amount of phosphorus standard (1 mg / mL), dilute it 24 times with standard diluent to obtain 1.292 mmol / L phosphorus standard; take 0.02 mL of phosphorus standard (1.292 mmol / L), add 0.48 mL of PiAssay buffer, mix thoroughly, and let stand at room temperature for 10 min; then centrifuge at 3000 g for 10 min, and collect the supernatant, which is the phosphorus standard working solution;

[0152] 3) Pi detection: Use a brand new enzyme-free sterile 96-well plate, set up blank wells, standard wells and test wells according to Table 3, and add the solution in sequence;

[0153] Table 3. Urinary phosphorus detection

[0154]

[0155] 4) Pi determination: Mix well, let stand at room temperature for 15 min, zero the instrument with the blank well, measure the absorbance of the standard tube and the test tube at 640 nm using an ELISA reader, and calculate the phosphorus concentration in the urine.

[0156] The calculation formula is: Phosphorus (mmol / L) = (A 测定 / A 标准 )×1.292×10;

[0157] The results of urine phosphorus test are as follows Figure 20 As shown in the figure, ** indicates P < 0.01, and *** indicates P < 0.001;

[0158] Depend on Figures 18-20 It can be seen that the urinary calcium level in the PC group was significantly higher than that in the FC group, while the urinary calcium level in the PH group was significantly lower than that in the PC group; there was no significant difference in urinary magnesium levels among the groups; the urinary phosphorus levels in the FH and PC groups were significantly higher than those in the FC group, while the urinary phosphorus levels in the PL and PH groups were significantly lower than those in the PC group.

[0159] Therefore, based on the measurement of mineral loss in urine, PM2.5 exposure affected the levels of some minerals in mice. Specifically, after PM2.5 exposure, mice lost more Ca and P in urine. Dandelion total flavonoids could reduce mineral loss, which is one of the reasons why they can improve bone density and bone quality.

[0160] Efficacy Verification: Effects of Total Flavonoids from Dandelion on Bone Structure at the Cellular Level

[0161] (I) Cell Culture

[0162] 1) Culture of pre-osteoblasts: Pre-osteoblasts (MC3T3-E1) were cultured in complete medium (α-MEM containing 10% FBS and 1% P / S) at 37°C in a carbon dioxide incubator (containing 5% CO2). When the cells reached 70-80% confluence, they were passaged. The specific method was to wash the cells once with PBS, add 0.25% trypsin for 2 min, and then immediately add twice the volume of complete medium to stop the digestion. After gently tapping the culture plate, the cells could be seen to become round and floating under an optical microscope. The cell suspension was collected and centrifuged at 1000 rpm at room temperature for 5 min. The cell pellet was resuspended in complete medium and passaged or plated at a ratio of 1:4.

[0163] 2) Culture of RAW264.7 cells: The cells were cultured in complete medium (α-MEM containing 10% FBS and 1% P / S) at 37°C in a carbon dioxide incubator (containing 5% CO2). When the cells reached 70-80% confluence, they were passaged. The specific method was to wash the cells once with PBS, add 0.25% trypsin to digest for 5 min, and then immediately add twice the volume of complete medium to stop the digestion. The adherent cells were gently blown off the wall with a pipette, and the cell suspension was collected and centrifuged at 1000 rpm at room temperature for 5 min. The cell pellet was resuspended in complete medium and passaged or plated at a ratio of 1:4-5.

[0164] (II) Preparation of Conditioned Culture Media

[0165] 1) Preparation of stock solutions: Under aseptic conditions, weigh 10 mg of PM2.5 standard particles into an EP tube and add 1 mL of α-MEM to prepare a stock solution of 10 mg / mL; prepare fresh for each use and sonicate before each use; weigh 0.064 g of total dandelion flavonoids and dissolve in 1 mL of anhydrous ethanol to prepare a stock solution of 64 mg / mL; weigh 0.0302 g of quercetin and dissolve in 4 mL of anhydrous ethanol to prepare a stock solution of 25 μmol / mL.

[0166] 2) Pre-treatment: After RAW264.7 cells grew to 70%, the blank control group and PM2.5 group were cultured in α-MEM containing 1% P / S for 12 hours; the low / medium / high dose dandelion total flavonoid groups were cultured in α-MEM containing 1% P / S and 50 / 100 / 200 μg / mL dandelion total flavonoids for 12 hours; the low / medium / high dose quercetin groups were cultured in α-MEM containing 1% P / S and 25 / 50 / 75 μM quercetin for 12 hours.

[0167] 3) PM2.5 intervention: After the pre-drug administration was completed, the culture medium of the blank control group was replaced with α-MEM containing 1% P / S; the culture medium of the PM2.5 group was replaced with α-MEM containing 1% P / S and 100 μg / mL PM2.5; the culture medium of the low / medium / high dose dandelion total flavonoid groups was replaced with α-MEM containing 1% P / S and 100 μg / mL PM2.5 and 50 / 100 / 200 μg / mL dandelion total flavonoids, respectively; the culture medium of the low / medium / high dose quercetin groups was replaced with α-MEM containing 1% P / S and 100 μg / mL PM2.5 and 25 / 50 / 75 μM quercetin, respectively. After the cells in each group were cultured for another 24 hours, the culture medium was collected, filtered through a 0.22 μm sieve, aliquoted, and stored at -80℃ for later use.

[0168] (III) Conditioned Culture Medium Intervention

[0169] 1) Conditioned culture medium intervention with total flavonoids from dandelion

[0170] After the MC3T3-E1 cells grew to 70%, the culture medium was changed, and they were cultured for another 2 days in complete medium containing 20% ​​conditioned medium. After the intervention, the cells were collected for subsequent detection. The conditioned medium was obtained from RAW264.7 cells in the blank control group, PM2.5 group, and low / medium / high dose dandelion total flavonoid groups.

[0171] 2) Quercetin conditioned medium intervention

[0172] After the MC3T3-E1 cells grew to 70%, the culture medium was changed and cultured for another 2 days in complete medium containing 20% ​​conditioned medium. After the intervention, the cells were collected for subsequent detection. The conditioned medium was obtained from RAW264.7 cells in the blank control group, PM2.5 group, and low / medium / high dose quercetin group.

[0173] (iv) RNA extraction and real-time quantitative PCR

[0174] 1) RNA extraction

[0175] (1) Collect the cells after the intervention into a sterile 1.5 mL centrifuge tube without enzymes, and immediately add 1 mL of RNAisoPlus Vertax to vigorously mix the cell sample and let it stand at room temperature for 10 min.

[0176] (2) Add 100 μL of 1-bromo-3-chloropropane (1 / 5 the volume of trizol) to each EP tube, invert the tubes to mix thoroughly, and let stand at room temperature for 5 min;

[0177] (3) Centrifuge at 12000 rcf, 4℃, for 15 min and collect 400 μL of the supernatant;

[0178] (4) Add 500 μL of isopropanol (1 / 2 the volume of trizol) to the supernatant of the EP tube, mix by pipetting, and let stand on ice for 10 min.

[0179] (5) Centrifugation: Centrifuge at 12000 rcf, 4℃, for 15 min and discard the supernatant in the EP tube;

[0180] (6) Add 1 mL of 75% alcohol (1 trizol volume) to each EP tube;

[0181] (7) Centrifugation: Centrifuge at 7500 rcf, 4℃, for 5 min, then discard the supernatant;

[0182] (8) Add 1 mL of 75% alcohol to the EP tube again, and centrifuge for the second time: 7500 rcf, 4℃, 5 min; discard the supernatant and remove the excess alcohol;

[0183] (9) Open the EP tube and place it in a fume hood for about 15 minutes. After it is completely dry, add 20 μL ddH2O to dissolve the RNA.

[0184] (10) RNA concentration and purity were determined using a NanoDrop2000 micro-volume spectrophotometer;

[0185] 2) RNA reverse transcription

[0186] Prepare a 20 μL reverse transcription system according to Table 4, and use an S1000 Thermal Cycle Pcr instrument to reverse transcribe RNA into cDNA. The reverse transcription program is 37℃ for 15 min, 85℃ for 5 sec, and store at 4℃. After reverse transcription, dilute the cDNA 5 times with ddH2O and store at -20℃.

[0187] Table 4 Reverse Transcription System

[0188]

[0189] 3) Real-time quantitative PCR (qRT-PCR)

[0190] Prepare a 10 μL real-time quantitative PCR reaction system according to Table 5. Add the reaction solution to a 384-well plate and perform qRT-PCR using a QuantStudio 7 Flex real-time quantitative PCR instrument. The amplification conditions are as follows: 95℃ for 10 min, 95℃ for 15 s, 60℃ for 1 min, with an upper limit of 40 cycles. The mRNA expression level of each gene relative to β-actin was calculated using the 2-ΔΔCt method. The primer sequences used are shown in Table 6.

[0191] Table 5 qRT-PCR reaction system

[0192]

[0193]

[0194] Table 6 qRT-PCR Primer Sequences

[0195]

[0196] (V) Protein extraction and immunoblotting

[0197] 1) Total protein extraction

[0198] (1) Collect cell samples after the intervention into enzyme-free sterile EP tubes and add 300 μL of protein lysis mixture (300 μL protein lysis mixture = 267 μL enhanced RIPA lysis buffer + 30 μL 10× phosphatase inhibitor + 3 μL 100× protease inhibitor); add 2 enzyme-free grinding beads (3 mm) to the centrifuge tube, homogenize using a multi-sample tissue homogenizer for 40 s, and then place the sample on ice and let it stand for 30 min;

[0199] (2) Centrifuge at 4℃ and 10000rcf for 10 min, transfer the protein supernatant to a new non-fermented EP tube and store on ice;

[0200] 2) Protein quantification and denaturation

[0201] (1) Based on the protein concentration detection method of the BCA kit, the 0.5 mg / mL protein standard was serially diluted according to Table 7:

[0202] Table 7. Serial dilution of protein standards

[0203]

[0204]

[0205] (2) Add 20 μL of protein standards of different concentrations and the protein to be tested to a 96-well plate, and then add 200 μL of BCA working solution (solution A:solution B = 50:1) to each well; after covering the plate, wrap the 96-well plate with aluminum foil and incubate it in a 37°C oven for 30 min. Then take it out and use a microplate reader to measure the absorbance at 562 nm. Calculate the concentration of the protein to be tested based on the standard line prepared by the protein standards.

[0206] (3) Dilute the protein sample to the same concentration (3ng / μL) using RIPA lysis buffer, then add one-quarter of the total volume of 5× Loading buffer, mix well, and place the EP tube in a metal bath at 100°C for 10 min to denature the protein; place the sample on ice to cool, and then freeze it in a -80°C freezer.

[0207] 3) Western blot assay for proteins

[0208] (1) Gel preparation: Wash and dry the glass plates and 10-hole comb, assemble the gel preparation tank, and prepare the separating gel and 5% concentrate according to Table 8; pour the separating gel between the two glass plates, press it flat with ddH2O, and pour off the ddH2O after the separating gel solidifies; then prepare the concentrate, gently add it to the top layer of the separating gel, insert the comb, and wait for the concentrate to solidify;

[0209] Table 8. Preparation of separating gel and stacking gel

[0210]

[0211] (2) Sample loading: Place the prepared gel along with the glass plate into the electrophoresis tank, add 1× electrophoresis solution until the stacking gel is submerged, remove the comb, add protein marker (2μL) and protein sample (10μL) into the well of the stacking gel, and add electrophoresis solution to the indicated position of the electrophoresis tank after the sample is added.

[0212] (3) Electrophoresis: Turn on the power supply to the electrophoresis tank, first electrophore at 50V for about 30 minutes (all samples are pressed into a straight line and located at the junction of the stacking gel and the separating gel), then pressurize to 100V until the protein is electrophoresed to the appropriate position and then stop electrophoresis; turn on the ice maker during electrophoresis to pre-cool the 1× transfer buffer.

[0213] (4) Transfer: Discard the electrophoresis buffer, prepare an 8cm long and 4.5cm wide PVDF membrane, and activate it by soaking it in methanol. Open the "sandwich" and immerse it completely in 1× transfer buffer. Remove the gel, cut off the stacking gel, and keep the separating gel. Assemble the transfer sandwich structure in 1× transfer buffer in the following order: (+) sponge - filter paper - methanol-activated PVDF membrane - separating gel - filter paper - sponge. After removing the air bubbles between the PVDF membrane and the gel, clamp the "sandwich" device, place the corresponding electrodes (black to black) into the transfer tank, add transfer buffer to the outer tank until the transfer tank indicator position is reached, close the lid, and transfer the membrane under constant voltage of 100V for 60-120min (the specific transfer time depends on the molecular weight of the target protein).

[0214] (5) Sealing: Prepare 5% skim milk. After the transfer is completed, take out the PVDF membrane and place it in a sealing box. Add 5% skim milk until it covers the surface of the PVDF membrane. Seale on a shaker at room temperature for 60 minutes.

[0215] (6) Primary antibody incubation: Dilute the primary antibody with 5% BSA according to the instructions, cut the PVDF membrane with the corresponding molecular weight according to the colorimetric marker, add the primary antibody, and incubate overnight (12-16h) in a shaker at 4℃.

[0216] (7) Secondary antibody incubation: Wash the membrane 4 times with 1×TBST, 5 min each time; then add the corresponding secondary antibody to the band (diluted with 1×TBST, the specific dilution ratio is according to the antibody instructions), and incubate at room temperature on a shaker for 1 hour;

[0217] (8) Development: Wash the membrane 4 times with 1×TBST, 5 min each time; prepare ECL developing solution, add freshly prepared ECL developing solution evenly to the band, and perform luminescence imaging using a chemical imaging system; save the required images, and perform statistical analysis of the protein bands using ImageJ software, and calculate the relative expression level of the target protein using GAPDH or HistoneH3 as internal reference proteins.

[0218] (VI) Results and Analysis

[0219] The effects of different concentrations of total dandelion flavonoids on the cell viability of RAW264.7 cells are as follows: Figure 21 As shown in the figure, * indicates P<0.05; the effects of different culture medium conditions with 20% concentration on the cell viability of MC3T3-E1 cells are as follows. Figure 22 As shown;

[0220] Depend on Figures 21-22 It can be seen that TF at concentrations below 200 μg / mL has no significant toxicity to RAW264.7 cells, and the 20% ratio of each conditional culture medium used also has no significant toxicity to MC3T3-E1 cells.

[0221] The expression levels of osteogenic differentiation-related genes BGLAP, SP7, ALP, COL1, and Bsp in MC3T3-E1 cells after intervention with different culture media were as follows: Figure 23 As shown, representative bands of osteogenic differentiation-related proteins BGLAP, SP7, ALP, and COL1 expressed in MC3T3-E1 cells after intervention with different culture media are shown in the figure. Figure 24 As shown; the protein expression levels of BGLAP, SP7, ALP, and COL1 are as follows: Figure 25 As shown in the figure, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001;

[0222] Depend on Figures 23-25 It was found that after intervention with 20% conditioned medium in MC3T3-E1 cells, PM2.5 conditioned medium significantly decreased the expression levels of osteogenic differentiation-related genes BGLAP, SP7, ALP, COL1, and Bsp in MC3T3-E1 cells. Low, medium, and high doses of TF conditioned medium significantly increased the gene expression level of ALP, medium and high doses of TF conditioned medium significantly increased the gene expression levels of Col1 and Bsp, and high doses of TF conditioned medium significantly increased the gene expression levels of BGLAP and SP7. Detection of osteogenic differentiation-related proteins using conventional Western blotting methods revealed that PM2.5 conditioned medium significantly decreased the expression levels of osteogenic differentiation-related proteins SP7, COL-1, and BGLAP in MC3T3-E1 cells, while low, medium, and high doses of TF conditioned medium significantly increased the expression level of COL-1 protein, and high doses of TF conditioned medium significantly increased the expression levels of SP7 and BGLAP protein.

[0223] The above results indicate that PM2.5 causes a decrease in the expression levels of osteogenic differentiation-related genes and proteins in pre-osteoblasts, while total flavonoids from dandelion can significantly improve the weakened osteogenic differentiation capacity caused by PM2.5 at both the gene and protein levels.

[0224] The expression levels of bone mineralization pathway-related genes Col16a1, Isg15, Clec3b, Ptn, and Matn1 in MC3T3-E1 cells after intervention with different culture media were as follows: Figures 26-30 As shown in the figure, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001;

[0225] Depend on Figures 26-30 It was found that PM2.5 conditioned medium significantly reduced the expression levels of genes related to the bone mineralization pathway, including Col16a1, Isg15, Clec3b, Ptn, and Matn1, while total flavonoids from dandelion could significantly improve the expression levels of these genes.

[0226] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. The application of total dandelion flavonoids in the preparation of products for preventing and treating osteoporosis, characterized in that, The extraction method for total flavonoids from dandelion includes the following steps: 1) Dry the whole dandelion plant, pulverize it, and obtain dandelion powder; 2) Mix the dandelion powder obtained in step 1) with 60% ethanol by volume, sonicate, and then filter. The ratio of dandelion powder to 60% ethanol by volume is 1:(25-35); 3) Mix the filter residue from step 2) with 40% ethanol by volume, heat under reflux, and then filter. The ratio of the filter residue to ethanol with a volume fraction of 40% is 1:(15-25); The heating reflux treatment is performed at a temperature of 55–65°C for 1–3 hours. 4) Combine the filtrates obtained in steps 2) and 3), concentrate and dry them to obtain the total dandelion flavonoids; The efficacy of the total flavonoids from dandelion was verified.

2. The application of total dandelion flavonoids according to claim 1 in the preparation of products for preventing and treating osteoporosis, characterized in that, In step 1), the drying process involves drying until the moisture content is less than 20%.

3. The application of total dandelion flavonoids according to claim 1 in the preparation of products for preventing and treating osteoporosis, characterized in that, In step 1), the crushing is to crush the material to pass through a 40-mesh sieve.

4. The application of total dandelion flavonoids according to claim 1 in the preparation of products for preventing and treating osteoporosis, characterized in that, In step 2), the ultrasonic treatment power is 120W, the temperature is 70-90℃, and the time is 25-35min.

5. The application of total dandelion flavonoids according to claim 1 in the preparation of products for preventing and treating osteoporosis, characterized in that, In step 4), the drying process involves drying until the total flavonoids of dandelion do not contain ethanol and the water content is less than 20%.

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