A kind of crystalline calcium oleoylserine and its preparation method and application

Abstract

The invention discloses a crystalline calcium oleoylserine and a preparation method and application thereof. The crystalline calcium oleoylserine of the invention has a better effect of preventing and treating osteoporosis than oleoylserine, magnesium oleoylserine and strontium oleoylserine. Compared with amorphous calcium oleoylserine, the crystalline calcium oleoylserine of the invention has a large tap density, a small angle of repose, good fluidity and strong compressibility, and is conducive to the preparation of a higher quality calcium oleoylserine preparation.

Description

Technical Field

[0001] The invention relates to the technical field of calcium oleoylserine, and in particular to crystalline calcium oleoylserine and a preparation method and application thereof. Background Art

[0002] WO2009125409 reports that oleoylserine has the effects of inhibiting bone resorption, promoting bone formation, and preventing and treating osteoporosis. The patent mentions salts of oleoylserine, but does not prepare specific oleoylserine salts, nor does it report the synthesis method and biological activity of specific oleoylserine salts.

[0003] The master's thesis of Zhejiang University, "Study on the Synthesis Process of Palmityl Ethanolamine and Oleoylserine and the Synthesis of Its Derivatives", believes that calcium, magnesium and strontium are important elements that promote human bone formation. Calcium and strontium salts of oleoylserine were prepared but no biological tests were conducted. In this paper, calcium and strontium salts of oleoylserine are prepared by first reacting oleoylserine with sodium hydroxide to prepare sodium oleoylserine, and then reacting with calcium chloride or strontium chloride to directly obtain calcium oleoylserine or strontium oleoylserine. There is no crystallization process, and the resulting product is an amorphous product. Amorphous oleoylserine salts have small particles, light weight, poor fluidity, and are difficult to prepare suitable pharmaceutical preparations. Summary of the invention

[0004] The purpose of the present invention is to provide a crystalline calcium oleoylserine and a preparation method and application thereof in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a crystalline calcium oleoylserine, wherein the crystalline calcium oleoylserine does not contain crystal water or any organic solvent.

[0007] Furthermore, the crystalline calcium oleoylserine has diffraction peaks at 5.6, 9.3, 18.6, 19.3, 20.7, 22.9, 23.2, 23.8, 29.3, and 37.6 in X-ray powder diffraction expressed in 2θ angles using Cu-Ka radiation.

[0008] Furthermore, the differential scanning calorimetry spectrum of the crystalline calcium oleoylserine has a single absorption peak at 172-182°C.

[0009] The second aspect of the present invention is to provide a method for preparing crystalline calcium oleoylserine, comprising the following steps:

[0010] Amorphous calcium oleoylserine is dissolved in an organic solvent and slowly cooled to 5-30°C, solid is precipitated, the solid is filtered out, and dried to obtain crystalline calcium oleoylserine.

[0011] Furthermore, the organic solvent is one or a mixture of acetic acid, propionic acid or butyric acid; the mass ratio of the amorphous calcium oleoylserine to the organic solvent is 1:(2-50).

[0012] Furthermore, the mass ratio of the amorphous calcium oleoylserine to the organic solvent is 1:(5-20).

[0013] The third aspect of the present invention is to provide the use of the above-mentioned crystalline calcium oleoylserine in the preparation of drugs for preventing and treating osteoporosis.

[0014] Furthermore, the drug for preventing and treating osteoporosis is in the form of tablets, coated tablets, capsules or granules.

[0015] Furthermore, the drug for preventing and treating osteoporosis also includes one or more of a disintegrant, a binder, and a diluent.

[0016] Furthermore, the disintegrant is one or a mixture of sodium starch acetate, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, and cross-linked polyvinyl pyrrolidone.

[0017] Furthermore, the adhesive is one or a mixture of starch, modified starch, sucrose, polyvinyl pyrrolidone, carboxymethyl cellulose, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.

[0018] Furthermore, the diluent is one or a mixture of starch, modified starch, microcrystalline cellulose, mannitol, and lactose.

[0019] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0020] The crystalline calcium oleoylserine of the present invention has better effects of preventing and treating osteoporosis than oleoylserine, magnesium oleoylserine and strontium oleoylserine.

[0021] Compared with amorphous calcium oleoylserine, the crystalline calcium oleoylserine of the present invention has a larger tap density, a smaller angle of repose, better fluidity and stronger compressibility, and is conducive to the preparation of a higher quality calcium oleoylserine preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the XRD pattern of crystalline calcium oleoylserine.

[0023] Figure 2 This is the XRD pattern of amorphous calcium oleoylserine.

[0024] Figure 3 This is the DSC spectrum of crystalline calcium oleoylserine.

[0025] Figure 4 This is the DSC spectrum of amorphous calcium oleoylserine.

[0026] Figure 5 This is the TG spectrum of crystalline calcium oleoylserine.

[0027] Figure 6 This is the TG spectrum of amorphous calcium oleoylserine.

[0028] Figure 7 This is a graph showing the results of the mouse bone marrow mesenchymal stem cell activity experiment in the present invention.

[0029] Figure 8 This is a graph showing the results of an alkaline phosphatase activity experiment after osteogenic induction in the present invention.

[0030] Fig. 9 This is a diagram showing the results of an Alizarin Red staining experiment after 21D osteogenic induction in the present invention.

[0031] Fig.10 This is a diagram showing the results of the Oil Red staining experiment after 14D adipogenesis induction in the present invention.

[0032] Fig.11 This is a graph showing the results of qRT-PCR analysis in the present invention.

[0033] Fig.12 This is the result diagram of Micro-CT in the present invention.

[0034] Fig.13 It is the Goldner trichrome staining diagram in the present invention. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0036] Example 1

[0037] This embodiment provides a method for preparing crystalline calcium oleoylserine, comprising the following steps:

[0038] Step 1, dissolve 2.7 mmol of oleoylserine (CAS No.: 107743-37-3) and solid sodium hydroxide in 15 mL of distilled water, mechanically stir at room temperature for 2 hours, and slowly drop 1.35 mmol of saturated calcium chloride aqueous solution when the reaction system becomes a colloidal state. Continue stirring for 2 hours after the dropwise addition, filter to obtain a white solid, wash the crude product twice with distilled water, and dry to obtain amorphous oleoylserine calcium (elemental analysis results: calcium content 5.18%, consistent with the theoretical content of 5.16%), with a yield of 96%;

[0039] Step 2: Mix 10 g of amorphous calcium oleoylserine (amorphous calcium oleoylserine) and 80 g of glacial acetic acid, stir, heat until dissolved, slowly cool to room temperature (20-30°C), and solid precipitates; filter out the solid by suction, and dry to obtain 8.92 g of crystalline calcium oleoylserine.

[0040] Example 2

[0041] This embodiment provides a method for preparing crystalline calcium oleoylserine, comprising the following steps:

[0042] Step 1, dissolve 2.7 mmol of oleoylserine (CAS No.: 107743-37-3) and solid sodium hydroxide in 15 mL of distilled water, mechanically stir at room temperature for 2 hours, and slowly drop 1.35 mmol of saturated calcium chloride aqueous solution when the reaction system becomes a colloidal state. Continue stirring for 2 hours after the dropwise addition, filter to obtain a white solid, wash the crude product twice with distilled water, and dry to obtain amorphous oleoylserine calcium (elemental analysis results: calcium content 5.18%, consistent with the theoretical content of 5.16%), with a yield of 96%;

[0043] Step 2: Mix 10 g of amorphous calcium oleoylserine and 100 g of propionic acid, stir, heat until dissolved, and slowly cool to room temperature to precipitate solid; filter out the solid by suction, and dry to obtain 9.24 g of crystalline calcium oleoylserine.

[0044] Example 3

[0045] This embodiment provides a method for preparing crystalline calcium oleoylserine, comprising the following steps:

[0046] Step 1, dissolve 2.7 mmol of oleoylserine (CAS No.: 107743-37-3) and solid sodium hydroxide in 15 mL of distilled water, mechanically stir at room temperature for 2 hours, and slowly drop 1.35 mmol of saturated calcium chloride aqueous solution when the reaction system becomes a colloidal state. Continue stirring for 2 hours after the dropwise addition, filter to obtain a white solid, wash the crude product twice with distilled water, and dry to obtain amorphous oleoylserine calcium (elemental analysis results: calcium content 5.18%, consistent with the theoretical content of 5.16%), with a yield of 96%;

[0047] Step 2: Mix 10 g of amorphous calcium oleoylserine and 150 g of butyric acid, stir, heat until dissolved, and slowly cool to room temperature to precipitate solid; filter out the solid by suction, and dry to obtain 8.32 g of crystalline calcium oleoylserine.

[0048] Comparative Example 1

[0049] This comparative example provides a method for preparing amorphous calcium oleoylserine: 2.7 mmol of oleoylserine (CAS No.: 107743-37-3) and solid sodium hydroxide are dissolved in 15 mL of distilled water, mechanically stirred at room temperature for 2 hours, and when the reaction system becomes a colloidal state, 1.35 mmol of a saturated calcium chloride aqueous solution is slowly added dropwise, and stirring is continued for 2 hours after the addition is completed, and filtered to obtain a white solid, and the crude product is washed twice with distilled water and dried to obtain the product calcium oleoylserine, with a yield of 96%. The elemental analysis result is: the calcium content is 5.18%, which is consistent with the theoretical content of 5.16%.

[0050] Comparative Example 2

[0051] This comparative example provides a method for preparing amorphous magnesium oleoylserine: 2.7 mmol of oleoylserine (CAS No.: 107743-37-3) and sodium hydroxide solid are dissolved in 15 mL of distilled water, mechanically stirred at room temperature for 2 hours, and when the reaction system becomes a colloidal state, 1.35 mmol of saturated magnesium chloride aqueous solution is slowly added dropwise, and stirring is continued for 2 hours after the addition, and filtered to obtain a white solid, and the crude product is washed twice with distilled water and dried to obtain the product magnesium oleoylserine, with a yield of 92%. The elemental analysis result is: the magnesium content is 3.17%, which is consistent with the theoretical content of 3.19%.

[0052] Comparative Example 3

[0053] This comparative example provides a method for preparing amorphous strontium oleoylserine: 2.7mmol of oleoylserine (CAS No.: 107743-37-3) and sodium hydroxide solid are dissolved in 15mL of distilled water, mechanically stirred at room temperature for 2h, and when the reaction system becomes a colloidal state, 1.35mmol of saturated strontium chloride aqueous solution is slowly added dropwise, and stirring is continued for 2h after the addition, and filtered to obtain a white solid, and the crude product is washed twice with distilled water and dried to obtain the product strontium oleoylserine, with a yield of 98%. The elemental analysis result is: the strontium element content is 9.80%, which is consistent with the theoretical content of 10.18%.

[0054] 1. Characterization of crystalline calcium oleoylserine and amorphous calcium oleoylserine

[0055] 1. X-ray powder diffraction (XRD) analysis:

[0056] (1) Instrument model: BRUKER D8 Advance X-ray diffractometer

[0057] X-ray: Identification X-ray, Cu-Ka radiation, wavelength 0.15406nm;

[0058] X-ray generator: 3Kw ceramic V-ray tube, working voltage 40KV, current 40mA;

[0059] Goniometer: θ-θ goniometer, radius 217.5mm;

[0060] Detector: LynxEye array detector;

[0061] Slot: Divergence slot is 1.0mm, anti-divergence slot is 8mm, receiving slot is 12.57mm, both Soller slots are 2.5°, NiKβ filter, no monochromator.

[0062] (2) Measurement conditions:

[0063] Ambient temperature: 20℃

[0064] Humidity: 50%RH

[0065] Scanning range: 3-90°

[0066] Scanning mode: continuous scanning, step length is 0.02°, scanning time per step is 0.1S;

[0067] Sample preparation method: Use a glass sample stage and adopt the normal loading method to fill the sample stage with the sample and compact it with flat glass.

[0068] The powder X-ray diffraction pattern of crystalline calcium oleoylserine is shown in Figure 1 The powder X-ray diffraction pattern of amorphous calcium oleoylserine is shown in Figure 2 .Depend on Figure 1 and Figure 2 By comparison, it can be seen that crystalline calcium oleoylserine has a diffraction absorption peak, while amorphous calcium oleoylserine has no diffraction absorption peak.

[0069] 2. Differential Scanning Calorimetry (DSC) Analysis:

[0070] (1) Instrument model: NETZSCH DSC200 PC

[0071] (2) Measurement conditions:

[0072] Ambient temperature: 18-22℃

[0073] Ambient humidity: 52%RH

[0074] Gas: N2, 40ml / min

[0075] Reference sample: а-Al2O3

[0076] Sample mass: 3.33 mg

[0077] Scanning range: 25-250℃

[0078] Heating rate: 10℃ / min

[0079] The DSC spectrum of crystalline calcium oleoylserine is shown in Figure 3 The DSC spectrum of amorphous calcium oleoylserine is shown in Figure 4 ,from Figure 3 and Figure 4 It can be seen that the initial melting temperature of crystalline calcium oleoylserine is 173.7℃, and the initial melting temperature of amorphous calcium oleoylserine is 168.1℃

[0080] 3. Thermogravimetric (TG) analysis:

[0081] (1) Instrument model: NETZSCH TG209 PC

[0082] (2) Measurement conditions:

[0083] Ambient temperature: 18-22℃

[0084] Ambient humidity: 52%RH

[0085] Gas: N2, 40ml / min

[0086] Reference sample: а-Al2O3

[0087] Sample mass: 7.4 mg

[0088] Scanning range: 25-800℃

[0089] Heating rate: 20°C / min.

[0090] The TG spectrum of crystalline calcium oleoylserine is shown in Figure 5 The TG spectrum of amorphous calcium oleoylserine is shown in Figure 6 .Depend on Figure 5 and Figure 6 It can be seen that both crystalline calcium oleoylserine and amorphous calcium oleoylserine do not contain crystal water or crystal solvent.

[0091] 4. Determination of bulk density, tap density and angle of repose

[0092] Bulk density, tap density and angle of repose are important indicators of material fluidity and compressibility.

[0093] Determined according to (0993 Bulk Density and Tap Density Determination Method of "Chinese Pharmacopoeia 2020 Edition Part 4").

[0094] Instrument: HR-2000 Powder Physical Properties Tester (Shandong Hengrui Instrument Co., Ltd.).

[0095] The results are shown in Table 1 below:

[0096] Table 1

[0097] Bulk density (g / ml) Tap density(g / ml) Angle of repose(℃) Crystalline calcium oleoylserine 0.67 0.73 29.3 Amorphous Calcium Oleoylserine 0.45 0.56 44.6

[0098] The results showed that crystalline calcium oleoylserine had better fluidity.

[0099] 2. Cell Experiment

[0100] In the following experiments, OS refers to oleoylserine, OS.Mg refers to magnesium oleoylserine, OS.Ca refers to crystalline calcium oleoylserine, and OS.Sr refers to strontium oleoylserine.

[0101] 1. Cell extraction and culture

[0102] The osteogenic induction kit and adipogenic induction kit were used to induce bone marrow stromal cells into osteoblasts and adipocytes. -12 M were added to the growth medium to evaluate their effects on osteogenic and adipogenic differentiation.

[0103] 2. Cell proliferation assay (24h; 48h; 72h)

[0104] The cultured BMSCs were seeded in 96-well culture plates (2*10 4 / well) and placed in a cell culture incubator for overnight culture. The cells seeded one day before were washed twice with PBS, and then 100ul of culture medium was added for intervention. At 24h, 48h, and 72h after intervention, the CCK-8 method was used to detect the changes in the proliferation ability of each group of cells: the old solution was discarded, and 90ul of culture medium and 10ul of CCK-8 were added to each 96-well plate to be tested, and incubated at 37℃ for 3h; the OD value of each experimental well at 450nm was measured using an enzyme marker.

[0105] The CCK-8 method was used to detect the effects of each drug on the -12 The effect of M concentration on BMSCs cell viability was analyzed from the results ( Figure 7 ) It can be seen that the cell proliferation after adding OS, OS.Mg, OS.Ca, and OS.Sr was significantly better than that in the blank group, indicating that each drug had no cytotoxicity and was beneficial to the proliferation of BMSCs.

[0106] 3. Osteogenesis Induction

[0107] Take BMSCs at 5*10 4 / cm 2 The cells were inoculated at a density of 100 μg / cm2 in the culture plate. After overnight adherence, the cells were observed to be 70%-80% confluent. The osteogenic induction medium was replaced and continued to be cultured. The medium was changed every 2 days. The osteogenic differentiation was identified by alkaline phosphatase activity test and Alizarin red staining test.

[0108] Each drug was divided into 10 -12M were added to the osteogenic induction medium to evaluate their effects on osteogenic differentiation. On days 3 and 7, the ALP activity of the OS.Ca group was significantly higher than that of the blank group and other drug groups ( Figure 8 ), indicating that the drug has a stronger ability to promote the osteogenic differentiation of BMSCs than other drugs. Alizarin red staining was performed 21 days after osteogenic induction to reflect the proliferation and differentiation of osteoblasts and the osteogenic potential of bone tissue. From the results ( Fig. 9 ) It can be clearly found that mineral deposition calcium nodules were produced in all drug groups, and OS.Ca had the best ability to form calcium nodules for the osteogenic differentiation of BMSCs.

[0109] 4. Adipogenesis Induction

[0110] Take BMSCs at 5*10 4 / cm 2 The cells were inoculated in a culture plate at a density of 100 μg / mL. After 12 h, the culture medium was changed to fat induction medium. At the same time, intervention drugs were added to each group according to the experimental design. The medium was changed every 3 days, and the formation of fat droplets was observed under an inverted microscope. On the 14th day after induction, the formation of fat droplets was identified by staining with Oil Red O. The cells were fixed with 4% paraformaldehyde solution at room temperature for about 30 minutes, stained with Oil Red O for 10 minutes, and the excess dye was washed off with PBS for photography. After taking pictures, 100 μL of isopropanol was added to each well to dissolve the Oil Red O dye in the stained cells, and quantitative analysis was performed using Image J software.

[0111] Each drug group was divided into 10 -12 M were added to the adipogenic induction medium to evaluate their effects on adipogenic differentiation ( Fig.10 ). Oil red staining under the microscope showed that when BMSCs were induced to adipogenesis for 14 days, the number of oil red staining positive cells in the blank control group was significantly higher than that in the drug group, and the OS.Ca group had smaller orange-red lipid droplets with full cytoplasm and larger gaps between lipid droplets than the OS group, indicating that the drug has the effect of inhibiting adipogenesis. Subsequently, the oil red staining area was quantitatively measured by Imagin J, and the results showed that the oil red staining area in the OS.Ca group was significantly lower than that in the OS group, OS.Mg group, OS.Sr group and blank group, indicating that the drug inhibited the adipogenic differentiation of BMSCs and was stronger than other drug groups.

[0112] 5. qRT-PCR

[0113] The cells in each group were collected, and total RNA was extracted according to the instructions of the Trizol kit and reverse transcribed into cDNA. The SYBRⅡ dye method RT-qPCR reaction was performed under the following conditions: 95℃, 30s; 95℃, 5s; 60℃, 30s, and 40 cycles. β-actin was used as an internal reference to determine the relative expression levels of β-catenin, Runx2, and ALP mRNA in cells.

[0114] To determine the molecular mechanism of OS.Ca's anti-osteoporosis effect, qRT-PCR analysis was performed to evaluate the mRNA levels of β-catenin and osteogenic genes (Alp, Runx2). Alp is synthesized by osteoblasts and reflects early osteogenic capacity, and Runx2 is an osteogenic differentiation transcription factor. Results ( Fig.11 ) showed that the mRNA of β-catenin and osteogenic genes Alp and Runx2 in BMSCs of the drug group was significantly enhanced. Therefore, we found that all the data showed that OS.Ca may play an anti-osteoporosis role through the osteogenic effect induced by β-catenin activation.

[0115] 3. Animal Experiments

[0116] In the following experiments, OS refers to oleoylserine, OS.Mg refers to magnesium oleoylserine, OS.Ca refers to crystalline calcium oleoylserine, and OS.Sr refers to strontium oleoylserine.

[0117] Grouping:

[0118] 1) Magnesium oleoylserine 0.1, 0.5, 1.0, 5.0, 10 mg / Kg / d

[0119] 2) Crystalline calcium oleoylserine 0.1, 0.5, 1.0, 5.0, 10 mg / Kg / d

[0120] 3) Strontium oleoylserine 0.1, 0.5, 1.0, 5.0, 10 mg / Kg / d

[0121] 4) Oleoylserine (OS) 3.0 mg / Kg / d

[0122] 5) Ovariectomized group (OVX)

[0123] 6) Sham group

[0124] Requirements: Female mice (20-30g)

[0125] Dosage (mg / kg / day) 0.1 0.5 1.0 5.0 10.0 Mouse 5 5 5 5 5

[0126] 1. Animals and Experimental Protocol

[0127] Eight-week-old female C57BL / 6J mice were provided by the Animal Center of Ningbo University. Mice underwent bilateral OVX or sham OVX, and the osteoporosis model was established by the classic bilateral ovariectomy method. Each mouse was injected with sodium pentobarbital intraperitoneally, the back skin was prepared, and a longitudinal incision (less than 1 cm) was made on the dorsal abdomen. The skin and abdominal muscles were then cut to find one side of the reddish-brown cauliflower-like ovary. The ovary was cut and removed at the oviduct, and the abdominal muscles and skin were sutured. The other ovary was operated in the same way. The mice were divided into 6 groups by the safety randomization method. After surgery, the mice were intraperitoneally injected with OS, calcium oleoylserine, magnesium oleoylserine, strontium oleoylserine, or balanced solution (ethanol / Tween 60 / normal saline (1:1:18)) every day for 6 weeks. Based on the recognized efficacy of OS in this model, the following in vivo doses were used: OS: 3 mg / kg / d; calcium oleoylserine, magnesium oleoylserine, strontium oleoylserine: 0.1, 0.5, 1, 5, 10 mg / kg / d. Mice were euthanized by cervical dislocation under anesthesia. After euthanasia, femurs were isolated, cleaned, fixed with 4% paraformaldehyde (pH 7.2) for 48 hours, and then stored in 70% ethanol until further use.

[0128] 2. CT detection and analysis

[0129] Before histological processing, femoral samples separated from each group were scanned using Micro-CT with a resolution of 10 μm. The three-dimensional structure of the femur was reconstructed using NRecon software. Morphological analysis, including bone mineral density (BMD), bone volume fraction (BV / TV, %), average trabecular thickness (Tb.Th, mm), average trabecular number (Tb.N, 1 / mm) and average trabecular separation (Tb.Sp, mm), was performed.

[0130] To evaluate the effects of OS and its derivatives on bone mass, an osteoporosis model was established in C57BL / 6J mice by OVX surgery. No adverse side effects or negative effects on survival were found during the entire intervention. Micro-CT results ( Fig.12 ) showed that OVX mice had significant bone loss compared with the sham-operated mice, indicating that the surgery successfully induced osteoporosis. At the same time, OS and calcium oleoylserine effectively reduced femoral bone loss and improved bone microstructure, as evidenced by the increase in BMD, BV / TV, Tb.Th and Tb.N and the decrease in Tb.Sp. Among them, BV / TV represents the ratio of bone tissue volume to tissue volume, which can directly reflect the changes in bone mass. Compared with OS, OS.Mg, and OS.Sr, OS.Ca has a stronger anti-osteoporosis effect; its bone mass even exceeds that of the sham-operated group, which has the effect of curing osteoporosis.

[0131] 3. Histological section staining

[0132] The left femur tissue was processed into 3 μm paraffin sections, and the morphological analysis was performed using Goldner trichrome-stained sections.

[0133] Goldern trichrome staining indicates ( Fig.13 ), OVX mice had sparse trabeculae and a large amount of fat accumulation below the growth plate, indicating an imbalance between osteogenesis and adipogenesis in OVX mice. After 6 weeks of OS and OS.Ca treatment, the area of ​​trabeculae increased significantly, while the area and diameter of fat droplets were greatly reduced.

[0134] in conclusion:

[0135] (1) Excellent osteogenic differentiation effect:

[0136] Quantitative analysis was performed by alkaline phosphatase activity assay. On the 3rd and 7th days, the ALP activity of the calcium oleoylserine group was higher than that of the oleoylserine group, indicating that the drug promoted the osteogenic differentiation of bone marrow mesenchymal stem cells more strongly than oleoylserine. This was also confirmed by the alizarin red staining experiment on the 21st day.

[0137] (2) Excellent inhibitory effect on adipogenic differentiation:

[0138] On the 14th day, oil red staining was performed, and red lipid particles were observed in the cytoplasm under the microscope. Compared with the other drug groups, the number of adipocytes in the calcium oleoylserine group decreased, indicating that the drug has a better effect in inhibiting adipogenic differentiation. Subsequently, the oil red staining area was quantitatively measured by Imagin J, and the results showed that the oil red staining area in the calcium oleoylserine group was significantly lower than that in the blank group and the other drug groups, indicating that the drug inhibited the adipogenic differentiation of bone marrow mesenchymal stem cells and was stronger than oleoylserine, magnesium oleoylserine, and strontium oleoylthreonate.

[0139] (3) Effectively rescue bone loss in osteoporotic mice:

[0140] Through mouse animal experiments, Micro-CT showed that OS and calcium oleoylserine effectively reduced femoral bone loss and improved bone microstructure, as evidenced by the increase in BMD, BV / TV, Tb.Th and Tb.N and the decrease in Tb.Sp. Compared with oleoylserine, magnesium oleoylserine and strontium oleoylserine, calcium oleoylserine had a better therapeutic effect.

[0141] (4) Goldner trichrome staining showed that the trabeculae of OVX mice were sparse and a large amount of fat accumulated under the growth plate, indicating an imbalance between osteogenesis and adipogenesis in OVX mice. Compared with the OS group, the trabecular area of ​​the OS.Ca group was significantly increased, and the area and diameter of the lipid droplets were significantly reduced. This indicates the synergistic effect of OS and calcium. (5) qRT-PCR results showed that the mRNA expression of β-catenin and osteogenic genes Alp and Runx2 in bone marrow mesenchymal stem cells in the drug group was significantly enhanced. Compared with the blank group, the OS.Ca group could significantly restore the inhibition of β-catenin signaling, up-regulated β-catenin expression, and increased Alp and Runx2 expression. Therefore, OS.Ca may exert its anti-osteoporosis effect by inducing osteogenesis through the activation of β-catenin signaling.

[0142] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A crystalline calcium oleoylserine, characterized in that: The crystalline calcium oleoylserine does not contain crystal water or any organic solvent; the crystalline calcium oleoylserine has diffraction peaks at 5.6, 9.3, 18.6, 19.3, 20.7, 22.9, 23.2, 23.8, 29.3, and 37.6 in X-ray powder diffraction expressed in 2θ angles using Cu-Ka radiation.

2. The crystalline calcium oleoylserine according to claim 1, characterized in that The differential scanning calorimetry spectrum of the crystalline calcium oleoylserine has a single absorption peak at 172-182°C.

3. A method for preparing crystalline calcium oleoylserine according to any one of claims 1 to 2, characterized in that: The steps include: Dissolving amorphous calcium oleoylserine in an organic solvent, slowly cooling to 5-30°C, solid precipitates, filtering out the solid, and drying to obtain crystalline calcium oleoylserine; Wherein, the organic solvent is one or a mixture of acetic acid, propionic acid or butyric acid; the mass ratio of the amorphous calcium oleoylserine to the organic solvent is 1:(2-50).

4. Use of the crystalline calcium oleoylserine according to any one of claims 1 to 2 in the preparation of a medicament for preventing and treating osteoporosis.

5. The use according to claim 4, characterized in that: The medicine for preventing and treating osteoporosis is in the form of tablets, capsules or granules.

6. The use according to claim 4, characterized in that: The medicine for preventing and treating osteoporosis is a coated tablet.

7. The use according to claim 4, characterized in that: The medicine for preventing and treating osteoporosis also includes one or more of a disintegrant, a binder, and a diluent.

Images