Application of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in preparing drugs for preventing and treating osteoporosis

Abstract

The present invention relates to the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester, and specifically discloses the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for treating and / or preventing osteoporotic diseases. The present invention also discloses a pharmaceutical composition for treating and / or preventing osteoporotic diseases comprising 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester. The present invention establishes a weightless osteoporosis mouse model to prove that 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester can improve the slow growth of femur caused by osteoporosis, improve the elasticity, strength and load-bearing capacity weakened caused by weightless osteoporosis; and can effectively promote the differentiation of osteoblasts.

Description

Technical Field

[0001] The invention relates to the technical field of biology and medicine, and in particular to the application of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of medicines for preventing and treating osteoporosis. Background Art

[0002] Osteoporosis is a metabolic bone disease characterized by decreased bone strength and increased risk of fractures. Osteoporosis is divided into two categories: primary and secondary. Primary osteoporosis is further divided into postmenopausal osteoporosis (type I), senile osteoporosis (type II) and idiopathic osteoporosis (including juvenile osteoporosis); while secondary osteoporosis is caused by other diseases.

[0003] Among them, senile osteoporosis is a common osteoporosis caused by the decrease of sex hormones in the body and physiological degeneration due to aging. It has become one of the important public health problems in the world.

[0004] Among secondary osteoporosis, long-term immobilization can also lead to osteoporosis. Among them, weightlessness osteoporosis is a special type of disuse osteoporosis caused by the weightlessness of spaceflight, and is also an urgent medical problem faced by astronauts in long-term spaceflight. It has similarities with the pathogenesis and countermeasures of senile osteoporosis. Therefore, the improvement and prevention of osteoporosis has become a hot topic in the research of people's livelihood health and aerospace medicine.

[0005] The applicant's laboratory previously cooperated with Chengdu Medical College to synthesize 3-hydroxy-3',4'-dihydroxy-butyric acid phenylethyl ester, the structure of which is shown in formula (I).

[0006]

[0007] In this application, the applicant used it in the relevant research on osteoporosis. The research results suggest that the compound can not only effectively promote osteoblast differentiation, but also improve the slow growth of femur caused by osteoporosis, and improve the weakened bone elasticity, strength and bearing capacity caused by weightlessness osteoporosis in mice. Therefore, it has application potential in the development of health foods and drugs for improving osteoporosis, promoting bone growth and treating fracture types. Summary of the invention

[0008] In view of the above background technology, the present invention provides the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for treating and / or preventing osteoporosis.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] The first aspect of the present invention provides the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for treating and / or preventing osteoporosis, wherein the 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester has a structure shown in formula (I),

[0011]

[0012] As a preferred embodiment, the osteoporosis disease is senile osteoporosis, immobility osteoporosis or weightlessness osteoporosis, preferably weightlessness osteoporosis.

[0013] The second aspect of the present invention provides the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for promoting bone formation.

[0014] The third aspect of the present invention provides the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for promoting osteoblast differentiation and / or calcification.

[0015] The fourth aspect of the present invention provides the use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of drugs for improving bone toughness, bone elasticity, bone strength and preventing and treating osteoporotic fractures.

[0016] The fifth aspect of the present invention provides a pharmaceutical composition for treating and / or preventing osteoporosis, which comprises a compound having a structure as shown in formula (I), an optically pure isomer of the compound, a mixture of enantiomers in any proportion, or a pharmaceutically acceptable salt thereof.

[0017]

[0018] Preferably, it further comprises one or more pharmaceutically acceptable carriers.

[0019] As a preferred embodiment, the effective intake concentration of the compound represented by the structure of formula (I) is 40 mg per day per kilogram of body weight.

[0020] The above technical solution has the following advantages or beneficial effects:

[0021] (1) It was first discovered that the compound 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester can improve the slow femoral growth caused by osteoporosis, suggesting that this compound may be able to promote bone growth.

[0022] (2) It was discovered for the first time that the compound 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester can effectively improve the weakened bone elasticity, strength and load-bearing capacity caused by osteoporosis, suggesting that this compound may be able to increase the organic matter content in bones, improve bone toughness, and thus prevent and treat fractures.

[0023] (3) It was discovered for the first time that the compound 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester can effectively promote the differentiation and calcification deposition of osteoblasts in an osteoporosis model, suggesting that it may have the effect of promoting bone formation and bone matrix mineralization, and improving bone loss caused by osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the femur length measurement method in Example 1.

[0025] Figure 2 This is a diagram of the femur length measurement results in Example 1 (CON is the normal control group, HLS is the tail-suspension experimental group, and HLS-HTHB is the tail-suspension plus 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester intervention group).

[0026] Figure 3 It is a graph of the experimental results of the femoral three-point bending mechanical properties test in Example 2 (Figure A is the elastic modulus; Figure B is the bending energy; Figure C is the ultimate stress; CON is the normal control group, HLS is the tail-suspension experimental group, and HLS-HTHB is the tail-suspension plus 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester intervention group; the ordinate is the maximum load).

[0027] Figure 4 It is the result of Alizarin Red S staining in Example 3 (CON is the normal control group, HLS is the tail suspension experimental group, and HLS-HTHB is the tail suspension plus 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester intervention group). DETAILED DESCRIPTION

[0028] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0029] Construction of weightlessness-induced osteoporosis model mice:

[0030] Among them, mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0031] 1. Experimental Materials

[0032] The applicant commissioned Chengdu Medical College to synthesize and prepare the compound 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester.

[0033] 2. Experimental Animal Breeding and Model Establishment

[0034] This experiment used 8-week-old healthy C57 male mice C57BL / 6 male mice, weighing about 22g. The mice were kept in an animal room with controlled temperature (22°C to 28°C) and humidity (60%). The lights in the room were maintained in a 12-hour day and 12-hour night cycle. During the experiment, the mice were able to eat and drink freely. This experiment used the tail suspension method to establish an osteoporosis model. The experimental mice were divided into 3 groups: (1) normal control group (CON group): gavage with normal saline every day, number of mice n = 14; (2) tail suspension experimental group (HLS group): osteoporosis model established by tail suspension method and gavage with normal saline every day, number of mice n = 9; (3) tail suspension plus 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester intervention group (HLS-HTHB group): tail suspension and gavage with 40 mg / kg 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester every day, number of mice n = 12, the detailed preparation method of the drugs is as follows:

[0035]

[0036] After one week of pre-adaptation in the animal room, mice were tail-suspended and given drug intervention at the same time. The tail-suspended cage was the original cage.

[0037] The steps of tail suspension are as follows: (1) fix the mouse and expose the tail; (2) disinfect and clean the tail with alcohol; (3) apply a small amount of rosin powder to increase the friction of the tail; (4) gently wrap the tail with pressure-sensitive tape three times, wrap it with medical cotton cloth, and wrap the tail with pressure-sensitive tape again; (5) step back in the middle of the tail and tie the tail with a thin rope; (6) hang the mouse on the metal frame of the cage, ensuring that the hind limbs are at a 30-degree angle to the horizontal plane; (7) place food and water in the cage for the mouse to ingest freely.

[0038] test:

[0039] 1. Femur length detection

[0040] The femur length measurement method is as follows: after tail suspension and 4 weeks of intervention, the mice are anesthetized and dissected, the femurs of the hind limbs are taken, and the lengths of both ends of the femurs are measured with a vernier caliper, such as Figure 1 shown.

[0041] 2. Three-point bending mechanical properties test

[0042] Take the mouse femur, cut off the muscle tissue attached to the bone, and place it on two supporting points with a spacing of 10mm. Apply a downward load to the femur above the midpoint of the two supporting points at a downward speed of 1.2mm / min. When the three contact points of the femur form two equal moments, three-point bending occurs, and the femur will break at the midpoint. Record the maximum load force required for each femur to break and the change in load force as the load displacement increases during the process. Measure the inner and outer diameters of the fracture cross section and calculate the femur elastic modulus, bending energy, ultimate stress and other indicators.

[0043] 3. Alizarin Red S staining experiment

[0044] Three groups of primary osteoblasts from mice were isolated and cultured in vitro. After 21 days of induction of differentiation, Alizarin Red S staining was performed to observe the osteogenic differentiation and calcium deposition of the cells. The specific staining method is as follows: (1) prepare 2% Alizarin Red S staining solution, shake evenly, and adjust the pH to 4.2; (2) remove the cells, remove the culture medium, and wash three times with PBS; (3) fix the cells with 4% paraformaldehyde for 30 minutes, and remove the paraformaldehyde; (4) wash three times with PBS; (5) add Alizarin Red S staining solution, stain at 37℃ for 30 minutes, and remove the staining solution; (6) wash with first-grade water several times to remove the residual staining solution; (7) observe and scan the results.

[0045] Statistical analysis

[0046] In the following examples, the CON group is the statistical result of 14 mice, the HLS group is the statistical result of 9 mice, and the HLS-HTHB group is the statistical result of 12 mice. The statistical results in the following examples are expressed in the form of Mean±SEM, and the data were analyzed using the One Way-ANOVA analysis method, p<0.001, which is statistically significant.

[0047] Example 1

[0048] This example demonstrates the improving effect of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester on delayed femoral development under weightlessness conditions.

[0049] Femoral length is an important indicator of bone growth and can reflect the growth and development of bones. In this embodiment, the length between the two ends of the femur is measured (see the measurement method for details). Figure 1 ), the results are shown in Figure 2: Compared with the CON group, the femur length of the HLS group was shortened, indicating that tail suspension affects femur development and growth, while the femur length of the HLS-HTHB group increased significantly after intervention with 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester. The results of this experiment prove that the compound 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester improves the femur length of weightless mice, indicating that the compound can promote bone development.

[0050] Example 2

[0051] This example demonstrates the effect of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester on the mechanical properties of the femur of mice under weightlessness conditions.

[0052] This embodiment adopts a three-point bending mechanical property test to detect the improvement effect of 3-hydroxy-3',4'-dihydroxy-butyric acid phenylethyl ester on the mechanical properties of mouse femur. The three-point bending mechanical property experiment is a classic indicator for detecting the elasticity, toughness and strength of materials. In this embodiment, the span of the three-point bending test is 10mm, and the loading rate is 1.2mm / min. Before the experiment, the grouping and micro-mechanical testing machine parameters are set, and the mouse femur is fixed at the same time to ensure that the placement position and direction of each sample are consistent as much as possible. Start the instrument, take the load force above the sample just breaking the femur as the test endpoint, and record the data.

[0053] Figure 3 The results of this experiment are statistically analyzed. Figure A is the elastic modulus, which is an important performance parameter to measure the ability of bones to resist elastic deformation. The larger the elastic modulus, the better the elasticity of the bones in response to external forces. Figure B is the bending energy. The bending energy index reflects the bending energy accumulated by the unit width of the bone to reach the limit span under the action of external forces. It is an important indicator for detecting the bending resistance of bones. The larger the bending energy, the stronger the bending resistance and the better the toughness. Figure C is the ultimate stress. The ultimate stress is the maximum stress that bones can withstand, that is, the bone strength. The larger the ultimate stress, the better the bone strength.

[0054] from Figure 3 It can be seen that after 4 weeks of tail suspension, the elastic modulus, bending energy, and ultimate stress of the HLS group decreased significantly compared with the CON group, while in the HLS-HTHB group, after the intervention of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester, the elastic modulus, bending energy, and ultimate stress of the femoral head of mice were greatly improved, especially the bending energy. The bending resistance of the HLS-HTHB group was even better than that of the CON group. Therefore, 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester has the effect of improving the weakening of elasticity, strength, and bearing capacity caused by weightlessness-induced osteoporosis.

[0055] Example 3

[0056] This example demonstrates the improving effect of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester on the differentiation ability of mouse primary osteoblasts.

[0057] Alizarin Red S calcium staining is a method that aims to generate a complex between calcium ions and Alizarin Red S through chelation technology to identify the calcium salt components of tissue cells. Calcium salt changes are one of the signs of osteoblast proliferation and differentiation and osteogenic potential of bone tissue. Alizarin Red S is an authoritative and classic technical method for analyzing the orange-red calcium deposition phenomenon in fixed cell samples, and is widely used in the study of bone cell or tissue pathophysiology.

[0058] In this example, osteoblast differentiation was induced for 21 days, and HTHB was given at the same time, and the cells were finally collected and stained. Figure 4 It can be clearly seen that compared with the CON group, the cell differentiation ability of the HLS group was weakened, while in the HLS-HTHB group, after the intervention of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester, the cell differentiation ability was significantly restored. Therefore, 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester can effectively promote osteoblast differentiation in the osteoporosis model, suggesting that it may promote osteoblast calcification and improve calcium loss after osteoporosis.

[0059] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester in the preparation of a drug for treating and / or preventing osteoporosis, wherein: The 3-hydroxy-3',4'-dihydroxy-butyric acid phenethyl ester has a structure shown in formula (I), The osteoporosis disease is weightlessness osteoporosis.

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