A kind of xanthohumol derivative and its application

Abstract

The present invention discloses a xanthohumol derivative, the general structural formula of which is selected from one of the following structures: The xanthohumol derivative prepared by the present invention is shown by pharmacological experiments to promote the proliferation and differentiation of osteoblasts and has good anti-osteoporosis activity, especially Class C compounds have better activity and can be used to prepare anti-osteoporosis drugs.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a xanthohumol derivative and application thereof. Background Art

[0002] Osteoporosis is a common disease characterized by systemic damage to bone mass and microstructure, which leads to increased bone brittleness and thus fractures. Osteoporosis is mainly divided into two categories: primary osteoporosis and secondary osteoporosis. With the aging of the population, the incidence of osteoporosis is increasing, becoming another health problem that middle-aged and elderly people need to face in addition to diabetes and hypertension.

[0003] The cause of osteoporosis is the disruption of the dynamic balance between osteoblasts and osteoclasts, which leads to a decrease in bone mass and even destruction of bone microstructure, resulting in a decrease in bone mass and bone density throughout the body. At present, the drugs commonly used in clinical practice for the prevention and treatment of osteoporosis can be divided into bone resorption inhibitors, bone mineralizers and bone formation promoters, but long-term use can cause various side effects, such as atypical femoral osteonecrosis, venous vascular embolism and stroke. Traditional Chinese medicine often exerts its therapeutic effect through multiple pathways and multiple targets, and has fewer adverse reactions. Compared with chemical drugs, it is more suitable for long-term use. A systematic literature survey found that xanthohumol has certain anti-osteoporosis activity, which can significantly promote the proliferation of osteoblasts and increase the ALP activity of osteoblasts, from early proliferation and differentiation to terminal bone matrix mineralization. Further studies have shown that xanthohumol can activate Runx2 expression and induce osteoblast differentiation through the p38 MAPK and ERK signaling pathways; it can also destroy the binding of RANKL and TRAF6 and inhibit the expression of ERK, which is very worth exploring in the anti-osteoporosis aspect. Summary of the invention

[0004] The object of the present invention is to provide a xanthohumol derivative.

[0005] The second object of the present invention is to provide a use of the xanthohumol derivative in the preparation of a drug for treating osteoporosis.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a xanthohumol derivative, the general structural formula of which is selected from one of the following structures:

[0008]

[0009] in:

[0010] R1 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0011] R2 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine), OH, CF3;

[0012] R3 is selected from hydrogen, methoxy, ethoxy, CF3;

[0013] R1 is hydrogen and R3 is methoxy; R1, R2, and R3 are not hydrogen at the same time;

[0014] R4 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0015] R5 is selected from halogen (fluorine, chlorine, bromine, iodine), OH, CF3;

[0016] R6 is selected from hydrogen, methoxy, ethoxy;

[0017] R7 is selected from hydrogen, halogen (fluorine, chlorine, bromine, iodine);

[0018] R8 is selected from halogen (fluorine, chlorine, bromine, iodine), OH, CF3;

[0019] R9 is selected from hydrogen, methoxy, ethoxy.

[0020] Most preferably, the structure of the xanthohumol derivative is selected from one of the following structures:

[0021]

[0022] The second aspect of the present invention provides a use of the xanthohumol derivative in the preparation of a medicament for treating osteoporosis.

[0023] The xanthohumol derivative provided by the invention can promote the proliferation and differentiation of osteoblasts, has good anti-osteoporosis activity, and can be used as a drug for treating osteoporosis.

[0024] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0025] The xanthohumol derivatives prepared by the present invention are shown by pharmacological experiments that compounds B1, C1, C2 and C3 have significant MC3T3-E1 Subclone 14 cell proliferation activity at concentrations of 0.2 and 1 μM, and the activity of compound C3 is better than that of the control drug xanthohumol. Since the proliferation ability of osteoblasts directly reflects the strength of bone formation, compound C3 is beneficial to the formation of osteoblasts. At concentrations of 0.2, 1 and 5 μM, B2, C1 and C3 can all increase the activity of ALP to varying degrees, especially at a concentration of 5 μM, B2 and C3 can significantly increase the activity of ALP, promote the expression of osteoblast alkaline phosphatase, promote cell mineralization, and are beneficial to the differentiation of osteoblasts, so the prepared xanthohumol derivatives can be used to prepare anti-osteoporosis drugs. DETAILED DESCRIPTION

[0026] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0027] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions provided by the manufacturer.

[0028] Example 1

[0029] Preparation of Class A Xanthohumol Derivatives

[0030]

[0031]

[0032] (1) Preparation of intermediate 3-methoxymethoxy-4-methoxybenzaldehyde (compound 2 in the reaction formula)

[0033] Take a 100mL round-bottom flask, add 4-methoxy-3-hydroxybenzaldehyde (1.5g, 9.86mmol) and 20mL dichloromethane in turn, add N,N-diisopropylethylamine (1.9g, 14.8mmol) under ice bath, and then slowly add chloromethyl methyl ether (1.2g, 14.8mmol). After reacting for 1 hour, the reaction is slowly warmed to room temperature, and the reaction system is brown and transparent. TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction process, and the reaction is terminated after 3 hours. After the reaction is completed, the reaction is quenched with an appropriate amount of saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase is washed with water and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, and the organic solvent is evaporated under reduced pressure. A light yellow oil (1.56g) is obtained by silica gel column chromatography, and the yield is 77%.

[0034] (2) Preparation of intermediate 3-isopentenyl-2,4,6-trihydroxy-acetophenone (compound 4 in the reaction formula)

[0035] Take a 1L round-bottom flask and add a magnetic stirrer, add 2,4,6-trihydroxyacetophenone (20g, 119mmol) and 300mL tetrahydrofuran in turn, add anhydrous potassium carbonate (34.5g, 250mmol) under stirring, reflux for 10 minutes, add 3,3-dimethylallyl bromide (18.2g, 122.5mmol) in batches, and the reaction system is yellow and turbid. TLC (petroleum ether: ethyl acetate = 1:1) monitors the reaction process, and the reaction is terminated after 6 hours. After the reaction is completed, the organic solvent is evaporated under reduced pressure, the residue is dissolved in water, extracted with ethyl acetate, the organic phase is washed with water and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, the organic solvent is evaporated under reduced pressure, and recrystallized with appropriate amounts of petroleum ether and ethyl acetate to obtain a light yellow solid (5.4g), with a yield of 19%.

[0036] (3) Preparation of intermediate 4,6-dimethoxymethoxy-3-isopentenyl-2-hydroxy-acetophenone (compound 5 in the reaction formula)

[0037] Take a 250mL round-bottom flask and add a magnetic stirrer, add 3-isopentenyl-2,4,6-trihydroxy-acetophenone (5.4g, 22.9mmol) and 40mL dichloromethane in turn, add N,N-diisopropylethylamine (8.9g, 68.7mmol) under ice bath, and then slowly add chloromethyl methyl ether (5.5g, 68.7mmol). After reacting for 1 hour, the reaction is slowly warmed to room temperature, and the reaction system is brown and transparent. TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction process, and the reaction is terminated after 3 hours. After the reaction is completed, the reaction is quenched with an appropriate amount of saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase is washed with water and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, and the organic solvent is evaporated under reduced pressure. A light yellow oil (3.8g) is obtained by silica gel column chromatography, and the yield is 51%.

[0038] (4) Preparation of intermediate 4,6-dimethoxymethoxy-2-methoxy-3-isopentenylacetophenone (compound 6 in the reaction formula)

[0039] Take a 250mL round-bottom flask and add a magnetic stirrer, add 4,6-dimethoxymethoxy-3-isopentenyl-2-hydroxy-acetophenone (3.8g, 11.7mmol) and 30mL N,N-dimethylformamide in turn, stir at room temperature to completely dissolve, add anhydrous potassium carbonate (3.2g, 23.4mmol) and iodomethane (3.5g, 23.4mmol), react at room temperature, and the reaction system is yellow and turbid. TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction process, and the reaction is terminated after 10 hours. After the reaction is completed, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride solution in turn, dry with anhydrous sodium sulfate, filter, and remove the organic solvent under reduced pressure. After silica gel column chromatography, a light yellow oil (3.7g) is obtained with a yield of 93%.

[0040] (5) Preparation of intermediate 3,4',6'-trimethoxymethoxy-2',4-dimethoxy-3'-isopentenyl chalcone (compound 7 in the reaction formula)

[0041] Take a 100mL round-bottom flask and add a magnetic stirrer, add 4,6-dimethoxymethoxy-2-methoxy-3-isopentenylacetophenone (400mg, 1.2mmol) and 3-methoxymethoxy-4-methoxy-benzaldehyde (247mg, 1.25mmol) in sequence, cool to 0°C in an ice-water bath, add 2.4mL of water-ethanol solution, dissolve 960mg of potassium hydroxide in 2.4mL of water-ethanol solution and add to the reaction bottle. React at 0°C for 1 hour under argon protection, then slowly warm to room temperature, and the reaction system is yellow and turbid. The reaction was allowed to proceed overnight, and the progress of the reaction was monitored by TLC (dichloromethane:methanol=80:1). After the reaction was completed, the reactant was poured into ice water, and the pH was adjusted to less than 5 with concentrated hydrochloric acid. The product was extracted with dichloromethane, and the organic phase was washed with water and a saturated sodium chloride solution in sequence, dried over anhydrous sodium sulfate, filtered, and the organic solvent was evaporated under reduced pressure. The product was purified by TLC preparative plate to obtain a yellow oil (543 mg) with a yield of 87%.

[0042] (6) Preparation of xanthohumol derivative 2',4-dimethoxy-3,4',6'-trihydroxy-3'-isopentenyl chalcone (Compound A1)

[0043] Take a 100mL round-bottom flask, add 3,4',6'-trimethoxymethoxy-2',4-dimethoxy-3'-isopentenyl chalcone (523mg, 1.0mmol) and 19mL methanol, then add 4.0mL 3 molar hydrochloric acid, reflux, the reaction system becomes orange-yellow transparent. TLC (petroleum ether: ethyl acetate = 1:1) monitors the reaction progress, after reflux for 45 minutes, pour the reactant into ice water, neutralize the reaction with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride solution in turn, dry with anhydrous sodium sulfate, filter, remove the organic solvent under reduced pressure, and purify with thin layer preparation plate to obtain orange-red powder A1 (140mg), with a yield of 36%.

[0044] Example 2

[0045] Preparation of compound A2: Compound A2 was obtained by replacing compound 1 in Example 1 with 2-chloro-3-hydroxybenzaldehyde and proceeding in the same manner as Example 1.

[0046] Example 3

[0047] Preparation of compound A3: Replace compound 2 in Example 1 with 3-trifluoromethylbenzaldehyde, and proceed with the same procedures as Example 1 to obtain compound A3.

[0048] Example 4

[0049]

[0050] (1) Preparation of intermediate 3,4',6'-trimethoxymethoxy-4-methoxy-3'-isopentenyl-2'-hydroxychalcone (compound 8 in the reaction formula)

[0051] Take a 100mL round-bottom flask and add a magnetic stirrer, add 4,6-dimethoxymethoxy-3-isopentenyl-2-hydroxy-acetophenone (500mg, 1.5mmol) and 3-methoxymethoxy-4-methoxy-benzaldehyde (315mg, 1.6mmol) in sequence, add 3mL of water-ethanol solution under ice bath, dissolve 1.2g of potassium hydroxide in 3mL of water-ethanol solution and add it to the reaction bottle. React for 1 hour under argon protection, then slowly warm to room temperature, and the reaction system is yellow and turbid. React overnight, TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction progress, after the reaction is completed, pour the reactant into ice water, adjust the pH to less than 5 with concentrated hydrochloric acid, extract with dichloromethane, wash the organic phase with water and saturated sodium chloride solution in sequence, dry with anhydrous sodium sulfate, filter, remove the organic solvent under reduced pressure, and purify by thin layer preparation plate to obtain a yellow oil (370mg), with a yield of 48%.

[0052] (2) Preparation of xanthohumol derivative 4-methoxy-2',3,4',6'-tetrahydroxy-3'-isopentenyl chalcone (Compound B1)

[0053] Take a 100mL round-bottom flask, add a magnetic stirrer, add 3,4',6'-trimethoxymethoxy-4-methoxy-3'-isopentenyl-2'-hydroxychalcone (370mg, 0.73mmol) and 14mL methanol, then add 3.0mL 3 molar hydrochloric acid, heat to reflux, and the reaction system becomes orange-yellow transparent. TLC (petroleum ether: ethyl acetate = 1:1) monitors the reaction process. After reflux for 45 minutes, pour the reactant into ice water, neutralize the reaction with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride solution in turn, dry with anhydrous sodium sulfate, filter, and remove the organic solvent under reduced pressure. Purify with a thin layer preparation plate to obtain an orange-red powder B1 (75mg), with a yield of 27%.

[0054] Example 5

[0055] Preparation of compound B2: Replace compound 2 in Example 4 with 2-chloro-3-methoxymethoxybenzaldehyde, and proceed with the same procedures as Example 4 to obtain compound B2.

[0056] Example 6

[0057] Preparation of compound B3: Replace compound 2 in Example 4 with 3-trifluoromethylbenzaldehyde, and proceed with the same procedures as Example 4 to obtain compound B3.

[0058] Example 7

[0059]

[0060] (1) Preparation of intermediate 4,6-dimethoxymethoxy-2-hydroxyacetophenone (compound 9 in the reaction formula)

[0061] Take a 500mL round-bottom flask and add a magnetic stirrer, add 2,4,6-trihydroxyacetophenone (8g, 47.6mmol) and 80mL dichloromethane in turn, add N,N-diisopropylethylamine (18.4g, 142.7mmol) under ice bath, and then slowly add chloromethyl methyl ether (11.5g, 142.7mmol). After reacting for 1 hour, the reaction is slowly warmed to room temperature, and the reaction system is brown and transparent. TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction process, and the reaction is terminated after 6 hours. After the reaction is completed, the reaction is quenched with an appropriate amount of saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase is washed with water and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, and the organic solvent is evaporated under reduced pressure. A light yellow oil (5.7g) is obtained by silica gel column chromatography, and the yield is 47%.

[0062] (2) Preparation of intermediate 4,6-dimethoxymethoxy-2-methoxyacetophenone (compound 10 in the reaction formula)

[0063] Take a 500mL round-bottom flask and add a magnetic stirrer, add 4,6-dimethoxymethoxy-2-hydroxy-acetophenone (5.7g, 22.2mmol) and 20mL N,N-dimethylformamide in turn, stir at room temperature to completely dissolve, add anhydrous potassium carbonate (6.1g, 44.4mmol) and iodomethane (6.3g, 44.4mmol), react at room temperature, and the reaction system is yellow and turbid. TLC (petroleum ether: ethyl acetate = 5:1) monitors the reaction process, and the reaction is terminated after 10 hours. After the reaction is completed, wash N,N-dimethylformamide with a small amount of water several times, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride solution in turn, dry with anhydrous sodium sulfate, filter, and evaporate the organic solvent under reduced pressure. After silica gel column chromatography, a light yellow oil (4.1g) is obtained with a yield of 68%.

[0064] (3) Preparation of intermediate 3,4',6'-trimethoxymethoxy-2',4-dimethoxy-chalcone (compound 11 in the reaction formula)

[0065] Take a 100mL round-bottom flask and add a magnetic stirrer. Add 4,6-dimethoxymethoxy-2-methoxyacetophenone (200mg, 0.74mmol) and 3-methoxymethoxy-4-methoxy-benzaldehyde (152mg, 0.78mmol) in turn. Add 1.5mL of water-ethanol solution under ice bath. Dissolve 592mg of potassium hydroxide in 1.5mL of water-ethanol solution and add it to the bottle. React for 1 hour under argon protection, then slowly warm to room temperature. The reaction system is yellow and turbid. TLC (petroleum ether: ethyl acetate = 3:1) monitors the reaction progress. The reaction is overnight. After the reaction is completed, the reactant is poured into ice water, the pH is adjusted to less than 5 with concentrated hydrochloric acid, and extracted with dichloromethane. The organic phase is washed with water and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, filtered, and the organic solvent is evaporated under reduced pressure. The light yellow oil (235mg) is purified by thin layer preparation plate, and the yield is 71%.

[0066] (4) Preparation of xanthohumol derivative 2',4-dimethoxy-3,4',6'-trihydroxychalcone (Compound C1)

[0067] Take a 100mL round-bottom flask, add 3,4',6'-trimethoxymethoxy-2',4'-dimethoxy-chalcone (235mg, 0.52mmol) and 10mL methanol, then add 2.1mL 3 molar hydrochloric acid, heat to reflux, the reaction system becomes orange-yellow transparent. TLC (petroleum ether: ethyl acetate = 1:2) monitors the reaction progress, after reflux for 45 minutes, pour the reactant into ice water, neutralize the reaction with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, wash the organic phase with water and saturated sodium chloride solution in turn, dry with anhydrous sodium sulfate, filter, remove the organic solvent under reduced pressure, and purify with a thin layer preparation plate to obtain orange-red powder C1 (132mg), with a yield of 81%.

[0068] Example 8

[0069] Preparation of compound C2: replace compound 2 in Example 7 with 2-chloro-3-methoxymethoxybenzaldehyde, and proceed with the same procedures as Example 7 to obtain compound C2.

[0070] Example 9

[0071] Preparation of compound C3: Replace compound 2 in Example 7 with 3-trifluoromethylbenzaldehyde, and proceed with the same procedures as Example 7 to obtain compound C3.

[0072] Table 1. Structure, yield and NMR data of the compounds prepared by the present invention

[0073]

[0074]

[0075]

[0076] Example 10

[0077] Pharmacological experiments of the compounds prepared in Examples 1 to 9 of the present invention

[0078] 1. Osteoblast proliferation assay of compounds

[0079] 1. Experimental method: The osteoblast proliferation experiment of the target compound was determined using the CCK-8 method.

[0080] 2. Cell selection: MC3T3-E1 Subclone 14 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0081] 3. Preparation of drugs and reagents:

[0082] Accurately weigh the sample to be tested, add it to a 1.5 mL centrifuge tube, add DMSO to make a 16 mM drug solution, and store it in a refrigerator at 4°C. Before use, take an appropriate amount and dilute it to 0.2 μM, 1 μM, or 5 μM with α-MEM complete medium containing 10% fetal bovine serum.

[0083] 4. Cell culture and passage:

[0084] MC3T3-E1 Subclone 14 cells were cultured in α-MEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO2. After the cells covered the bottom of the dish, the medium was aspirated, washed once with sterile PBS, and 1 mL of trypsin cell digestion solution was added to digest the cells for about 30 seconds. After slowly blowing the cells off completely, 5 mL of complete medium was added, and the cells were blown off with a pipette, centrifuged, and divided into 3 new cell culture dishes, and then 10 mL of complete medium was added and cultured continuously.

[0085] 5. Drug treatment:

[0086] The cells cultured to the logarithmic growth phase were collected and the cell concentration was adjusted to 1×10 cells / mL using α-MEM complete medium containing 10% fetal bovine serum. 4 / mL, add 100μL of cell suspension to each well of a 96-well cell culture plate. After culturing the culture plate in a CO2 incubator for 24 hours, remove the original culture medium, wash twice with sterile PBS, add 200μL of fresh culture medium containing 0.2μM, 1μM, and 5μM of each drug, set up 6 replicates for each concentration, and set up 6 replicates for the xanthohumol control group and the blank control group, and continue to culture in a CO2 incubator for 48 hours. Take out the culture plate, discard the culture medium, wash twice with an appropriate amount of PBS, add 100μL of serum-free α-MEM culture medium and 10μL CCK-8 to each well, continue incubation for 0.5 hours, use an enzyme reader to measure the absorbance value at a wavelength of 450nm, and calculate the proliferation activity of the target compound on osteoblasts.

[0087] Proliferation rate = (A450 drug-treated well / A450 blank control well) × 100%.

[0088] The effects of the target compounds on the proliferation rate of MC3T3-E1 Subclone 14 cells are shown in Table 2.

[0089] Table 2 Proliferation rate of MC3T3-E1 Subclone 14 cells at concentrations of 0.2 μM, 1 μM, and 5 μM (%)

[0090] Compound 0.2μM 1μM 5μM A1 96.1 93.7 99.3 A2 94.2 95.9 101.3 A3 97.9 97.8 95.3 B1 101.3 111.9 107.3 B2 83.1 82.0 90.3 B3 98.5 96.7 93.4 C1 101.7 103.9 107.3 C2 102.6 105.3 104.4 C3 107.5 118.7 138.6 Xanthohumol 102.3 105.9 97.2

[0091] Osteoblasts play a key role in the process of bone formation, and the proliferation ability of osteoblasts reflects the strength of bone formation. At a concentration of 0.2 μM, compounds B1, C1, C2 and C3 have proliferation activity, with proliferation rates of 101.3%, 101.7%, 102.6% and 107.5%, respectively, and the activities of compounds C2 and C3 are better than xanthohumol; at a concentration of 1 μM, compounds B1, C1, C2 and C3 also have proliferation activity, among which B1 and C3 have better activities than xanthohumol, with proliferation rates of 111.9% and 118.7%, respectively; at a concentration of 5 μM, compounds A2, B1, C1, C2 and C3 have significant proliferation activity, and the activity is higher than xanthohumol. Therefore, most of the target compounds have good proliferation activity on MC3T3-E1 Subclone 14 cells, which is conducive to the formation of osteoblasts, especially the C-type compounds, whose activity is better than the control drug xanthohumol, and C3 has the best activity, which can be used to prepare anti-osteoporosis drugs.

[0092] 2. Determination of osteoblast alkaline phosphatase (ALP) activity

[0093] 1. Experimental method: The alkaline phosphatase activity of osteoblasts was determined using an alkaline phosphatase kit.

[0094] 2. Cell selection: MC3T3-E1 Subclone 14 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0095] 3. Preparation of drugs and reagents:

[0096] Accurately weigh the sample to be tested, add it to a 1.5 mL centrifuge tube, add DMSO to make a 16 mM drug solution, and store it in a refrigerator at 4°C. Before use, take an appropriate amount of α-MEM complete medium containing 10% fetal bovine serum and dilute it to 0.2 μM, 1 μM, or 5 μM.

[0097] 4. Cell culture and passage:

[0098] MC3T3-E1 Subclone 14 cells were cultured in α-MEM complete medium containing 10% fetal bovine serum at 37°C and 5% CO2. After the cells covered the bottom of the dish, the medium was aspirated, washed once with sterile PBS, and 1 mL of trypsin cell digestion solution was added to digest the cells for about 30 seconds. After slowly blowing the cells off completely, 5 mL of complete medium was added, and the cells were blown off with a pipette, centrifuged, and divided into 3 new cell culture dishes, and then 10 mL of complete medium was added and cultured continuously.

[0099] 5. Drug treatment:

[0100] Move the well-growing MC3T3-E1 Subclone 14 cells to a clean bench, wash twice with PBS, add 1 mL of 0.25% trypsin digestion solution, and add 3 mL of complete culture medium to terminate the digestion after 1 minute. Centrifuge at 1000 rpm for 5 minutes and move to a sterile operating table, discard the supernatant, add an appropriate amount of culture medium, and repeatedly blow to resuspend. Count under a microscope and calculate 1×10 4 Each well was inoculated with 150 μL of PBS solution, and 200 μL of PBS solution was added to the outer circle of the 96-well plate to prevent the cell suspension from volatilizing. After 24 hours of culture, the culture medium was discarded, and the cells were washed twice with an appropriate amount of PBS. Then 100 μL of culture medium containing different drug concentrations (0.2 μM, 1 μM, 5 μM) was added, and a blank control group was set up. Each group was set up with 6 replicates and cultured for 48 hours. 30 μL of supernatant was aspirated from each well in a new 96-well plate according to the previous order, and the alkaline phosphatase (ALP) kit instructions were used for determination.

[0101] ALP activity (%) = ALP activity of experimental group / ALP activity of blank control group × 100%

[0102] The results of the target compound's effect on alkaline phosphatase activity in MC3T3-E1 Subclone 14 cells are shown in Table 3.

[0103] Table 3 Effects of the target on alkaline phosphatase activity in MC3T3-E1 Subclone 14 cells

[0104] Compound 0.2μM 1μM 5μM A1 98.0 97.0 96.4 A2 97.5 97.3 98.3 A3 96.3 97.7 97.3 B1 92.3 92.9 93.1 B2 104.2 107.7 116.0 B3 95.3 89.1 86.0 C1 102.2 102.4 103.1 C2 97.8 99.3 98.2 C3 102.6 104.1 112.7 Xanthohumol 102.0 103.0 103.6

[0105] Alkaline phosphatase is distributed in many tissues such as animal liver, bones and small intestine. ALP is a marker of osteoblast differentiation. Its activity can reflect the degree of differentiation of osteoblasts, so it is also an important indicator for evaluating bone formation. At concentrations of 0.2, 1 and 5 μM, B2, C1 and C3 can increase the activity of ALP to varying degrees. Especially at a concentration of 5 μM, B2 and C3 can significantly increase the activity of ALP, promote the expression of osteoblast alkaline phosphatase, promote cell mineralization, and facilitate the differentiation of osteoblasts. Among them, the C-type compound has the best activity, which is consistent with the results of the proliferation activity of osteoblasts, and can be used to prepare anti-osteoporosis drugs.

[0106] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A xanthohumol derivative, characterized in that: The structure of the xanthohumol derivative is selected from one of the following structures:

2. Use of the xanthohumol derivative according to claim 1 in the preparation of a drug for treating osteoporosis.

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