Gentiopicroside strontium complex and preparation method and application thereof

Abstract

The application discloses a preparation method of a strontium-gentiopicroside complex, which comprises the following steps: (1) dissolving gentiopicroside and strontium chloride in ddH2O respectively to prepare a clear and transparent gentiopicroside solution A and a strontium chloride solution B, and the molar concentration ratio of the solution A to the solution B is 1:2-6; (2) under continuous stirring, the solution A is added to the solution B at a speed of 60-120 drops / min, and after the addition is completed, a mixed solution C is obtained; (3) 1 / 2-2 / 3 of the volume of anhydrous ethanol is added to the mixed solution C, the temperature is increased to 40-60 DEG C under the protection of argon and in the dark, and after sufficient mixing, a solution D is obtained; (4) the solution D is cooled to room temperature, the ethanol is removed by rotary evaporation at a temperature of no more than 40 DEG C, and a viscous liquid E is obtained; and (5) the viscous liquid E is freeze-dried, recrystallized by using methanol, and white powder is obtained, which is the strontium-gentiopicroside complex. The gentiopicroside complexed with strontium has obvious bone repair effect, and has better stability and bioavailability.

Description

Technical Field

[0001] This invention relates to the field of compound complexation technology, specifically to a strontium gentiopicroside complex, its preparation method, and its application. Background Technology

[0002] Gentipicroside (GPS) possesses anti-inflammatory, immunomodulatory, and osteogenic effects. However, as an iridoid glycoside, its stability is poor and its bioavailability is low due to the influence of pH, temperature, moisture, and enzymes, thus limiting its biological activity. GPS is rapidly absorbed in mice, but its bioavailability remains low. Therefore, improving its stability, bioavailability, and duration of action is a prerequisite for it to exert its immunomodulatory and osteogenic functions.

[0003] Strontium ions are important physiological elements in the human body. Strontium plays an important role in bone formation. Strontium can promote the proliferation of osteoblasts and pluripotent stem cells and improve bone metabolism. However, free strontium ions are toxic in the body, which limits their clinical application in bone repair and other aspects.

[0004] How to eliminate the negative effects of both and enable them to work synergistically to better perform their functions such as bone repair is worthy of further research. Summary of the Invention

[0005] The purpose of this invention is to provide a gentiopicroside strontium complex, its preparation method and application. This gentiopicroside strontium complex has strong stability, high bioavailability, and can be used for bone repair.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] (1) Dissolve 0.1-0.3 mmol of gentiopicroside in 1.0 mL of ddH2O to obtain a clear and transparent solution A. Dissolve strontium chloride in 1.0 mL of ddH2O at a molar ratio of 1:2-6 to obtain a clear and transparent solution B.

[0008] (2) While stirring continuously, solution A is added dropwise to solution B. After the addition is complete, a mixed solution C is obtained.

[0009] (3) Add anhydrous ethanol to the mixture C, heat to 40-60℃ under argon protection and light protection, and mix thoroughly to obtain solution D;

[0010] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0011] (5) Freeze-dry the viscous liquid E and recrystallize it with methanol to obtain a white powder, which is the gentiopicroside strontium complex.

[0012] Furthermore, in step (2), the stirring speed is 500-800 rpm / min, and the stirring time is 2-6 hours.

[0013] Furthermore, the dripping rate in step (2) is 60-120 drops / min.

[0014] Furthermore, after the addition in step (2) is completed, 0.5-1.0 mL of ddH2O is added to ensure complete transfer.

[0015] Further, 10-30 mL of anhydrous ethanol is added in step (3).

[0016] Furthermore, in step (3), thorough mixing is achieved by stirring for 24-48 hours.

[0017] Furthermore, the freeze-drying in step (5) is carried out continuously for 24-48 hours at a vacuum of -80 to -40°C and 800-1000 Pa.

[0018] Furthermore, the recrystallization in step (5) is performed 2-4 times.

[0019] The gentiopicrin strontium complex prepared by the above-mentioned method contains strontium ions complexed on the hydroxyl and carbonyl groups of gentiopicrin, with the strontium ions accounting for 20-40% of the complex, and the particle size of the complex is smaller than that of the gentiopicrin monomer.

[0020] The above-mentioned gentiopicroside strontium complex is used in bone repair.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. Inspired by the theory of "mutual reinforcement" in traditional Chinese medicine, this invention complexes strontium ions with gentiopicroside through chemical bonds. The complexed gentiopicroside strontium has the same macrophage proliferation-promoting effect as the monomeric gentiopicroside and can be used for bone cell repair. Moreover, the complexed compound has a longer duration of action, and the stability and bioavailability of the complexed gentiopicroside are also stronger than those of the monomer.

[0023] 2. This invention avoids the toxicity of free strontium ions by complexing strontium ions with the hydroxyl and carbonyl groups of the macromolecule gentiopicroside. At the same time, the prepared gentiopicroside strontium complex also possesses the characteristics of both strontium and carbonyl groups, and enables strontium to be effectively released in vivo. This allows strontium to exert its effects on reducing inflammation and promoting osteoblast proliferation, thereby altering the immune environment of bone and promoting the proliferation and differentiation of bone repair-related cells. It plays an important role in bone repair by regulating bone immunity. Attached Figure Description

[0024] Figure 1The FTIR values ​​before and after the complexation of gentiopicrin and strontium in Example 1 of this invention;

[0025] Figure 2 This is a SEM image of gentiopicroside before it complexes with strontium in Example 1 of the present invention;

[0026] Figure 3 This is a SEM image of gentiopicroside complexed with strontium in Example 1 of the present invention.

[0027] Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the gentiopicroside strontium complex prepared in Example 1 of the present invention.

[0028] Figure 5 Thermogravimetric analysis (TGA) curve of the gentiopicroside strontium complex prepared in Example 1 of this invention;

[0029] Figure 6 This is a bar chart showing the effect of the gentiopicroside strontium complex and gentiopicroside monomer prepared in Example 1 of the present invention on cell proliferation. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0031] The preparation method of the gentiopicroside strontium complex of the present invention includes the following steps:

[0032] (1) Dissolve 0.1-0.3 mmol of gentiopicroside in 1.0 mL of ddH2O to obtain a clear and transparent solution A. Dissolve strontium chloride in 1.0 mL of ddH2O at a molar ratio of 1:2-6 to obtain a clear and transparent solution B.

[0033] (2) While stirring continuously, add solution A dropwise to solution B, then add 0.5-1 mL of ddH2O, and continue stirring for 2-6 hours until the solution is completely transferred to obtain mixed solution C;

[0034] (3) Add 10-30 mL of anhydrous ethanol to the mixture C, heat to 40-60 °C under argon protection and light protection, and mix thoroughly to obtain solution D;

[0035] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0036] (5) The viscous liquid E is freeze-dried at a temperature of -80 to -40°C and a vacuum of 800 to 1000 Pa for 24 to 48 hours, and then recrystallized with methanol 2 to 4 times to obtain a white powder, which is the gentiopicrin strontium complex.

[0037] Example 1: A method for preparing a gentiopicroside strontium complex, comprising the following steps:

[0038] (1) Dissolve 107 mg (0.3 mmol) gentiopicroside in 1.0 mL ddH2O to obtain a clear and transparent solution A, and dissolve 95.1 mg (0.6 mmol) strontium chloride in 1.0 mL ddH2O to obtain a clear and transparent solution B;

[0039] (2) While stirring continuously at a speed of 600 rpm / min, add solution A to solution B at a dropping rate of 80 drops / min, then add 0.5 mL of ddH2O, and continue stirring for 6 hours to completely transfer the solution, thus obtaining mixed solution C;

[0040] (3) Add 20 mL of anhydrous ethanol to the mixture C, heat to 60 °C under argon protection and light protection, and stir for 48 hours to obtain solution D;

[0041] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0042] (5) The viscous liquid E was freeze-dried at -80℃ and 1000Pa vacuum for 24 hours, and then recrystallized twice with methanol to obtain a white powder, which is the gentiopicrin strontium complex.

[0043] The obtained gentiopicroside strontium complex has the following characteristics:

[0044] 1. Structural characterization

[0045] 1.1 Infrared Spectroscopy (FTIR)

[0046] See Figure 1 After gentiopicrin complexes with strontium, the hydroxyl vibration peaks at wavenumbers of 3566 and 3493 shift to lower wavenumbers; the hydroxyl vibration peak at wavenumber 3288 weakens into a peak; the carbonyl vibration peak at wavenumber 1703 also shifts to lower wavenumbers and its intensity is significantly reduced; the absorption at wavenumber 598 is due to the strontium salt, indicating that the strontium ion complexes with the hydroxyl and carbonyl groups of gentiopicrin.

[0047] 1.2 Scanning Electron Microscopy (SEM)

[0048] See Figure 2 and Figure 3 Compared to GPS, GPS-Sr has a smaller particle size and a more uniform distribution because the addition of strontium helps to optimize the structure of GPS.

[0049] 1.3 Energy Dispersive Spectroscopy (EDS)

[0050] Referring to Tables 1 and 2 below, EDS analysis revealed that Sr ions accounted for 24.1% in the GPS-Sr complex, while no Sr ions were detected in the original GPS.

[0051] Table 1 GPS Energy Spectrum EDS Detection

[0052] element Line type Apparent concentration k ratio Wt% Wt%Sigma At% C K-line system 126.73 1.26728 59.30 0.25 66.01 O K-line system 120.00 0.40382 40.65 0.25 33.97 Cl K-line system 0.13 0.00115 0.05 0.05 0.02 Sr L-line system 0.00 0.00000 0.00 0.10 0.00 Total: 100.00 100.00

[0053] Table 2 GPS-Sr energy dispersive spectroscopy (EDS) data

[0054] element Line type Apparent concentration k ratio Wt% Wt%Sigma At% C K-line system 33.31 0.33311 23.47 0.22 64.29 O K-line system 3.81 0.03337 1.61 0.04 3.31 Cl K-line system 58.49 0.55785 19.54 0.26 18.13 Sr L-line system 63.91 0.55899 24.10 0.30 9.05 Au M-line system 56.74 0.56743 31.28 0.45 5.22 Total: 100.00 100.00

[0055] 2. Purity Analysis

[0056] The purity of the prepared gentiopicroside strontium complex was determined by high performance liquid chromatography (HPLC). The sample was dissolved in methanol to a final concentration of 1.0 mg / mL, and 5.0 μL was injected. Mobile phase A was acetonitrile, and mobile phase B was ddH₂O, both free of trifluoroacetic acid. The detection wavelength was 254 nm. (See [reference]). Figure 4 The final purity was approximately 96%.

[0057] 3. Stability Analysis

[0058] Thermogravimetric analysis (TGA) was used to test the stability of GPS and GPS-Sr. The TGA curves are shown below. Figure 5 As shown, weight loss below 172°C is likely due to physically adsorbed water and guest water, while weight loss between 172°C and 400°C is likely due to the decomposition of GPS and GPS-Sr. GPS-Sr is superior to GPS in terms of physical water adsorption. Compared to GPS, the weight loss of GPS-Sr is significantly reduced between 200°C and 600°C, which should be attributed to the complexation between GPS and Sr.

[0059] 4. Cell experiments

[0060] GPS-Sr complex and GPS were prepared into sample solutions with concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 500 μg / mL in DMEM1640 medium containing 5% FBS (fetal bovine serum), respectively. DMEM medium containing 5% FBS without the sample was used as a control solution.

[0061] Because RAW264.7 macrophages play an important role in regulating the immune environment of bone, and their differentiation and proliferation regulate osteocytes, mouse RAW264.7 macrophages were used for this study. Macrophages were scraped from the culture surface using a cell scraper and pipetted to prepare a single-cell suspension. Cells were then counted on a counting chamber. The single-cell suspension was diluted with DMEM medium containing 5% FBS and seeded into 96-well plates, with approximately 2000 cells per well. 100 μL of the diluted single-cell suspension was added to each well. The 96-well plates were incubated in a CO2 incubator for 24 hours. The culture medium was then aspirated from the 96-well plates, and the cells were then added to the wells of the 96-well plates. 200 μL of the above sample solution was added as the experimental group, and 200 μL of the control solution was added as the control experiment. Each concentration of sample solution was set up with 4 parallel wells. Gentianoside strontium complex and gentiopicroside were prepared into 8 sample solutions with concentrations of 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 500 μg / mL. Each concentration of sample solution was set up with 4 wells, and each well contained 200 μL of sample solution. After 5 days of incubation, the results were detected.

[0062] The proliferation rate of macrophages was detected using a cell counting kit-8. The principle of the cell counting kit-8 is that the component WST-8 is bioreduced by cellular dehydrogenases to a culture medium-soluble orange product, and the amount of product produced is directly proportional to the number of viable cells. The absorbance of each well was measured using a microplate reader to obtain the absorbance value, which reflects the cell proliferation rate. Figure 6 The results show that cells cultured in cell culture media containing GPS or GPS-Sr solutions at concentrations of 10 μg / mL to 100 μg / mL exhibited significantly greater proliferation than the control group. Furthermore, compared to the GPS group, the GPS-Sr group showed more significant cell proliferation at a concentration of 100 μg / mL. This indicates that the prepared gentiopicroside strontium complex has a more significant promoting effect on macrophage proliferation than the gentiopicroside monomer, suggesting that the gentiopicroside strontium complex has a better cell proliferation-promoting effect.

[0063] Example 2: A method for preparing a gentiopicroside strontium complex, comprising the following steps:

[0064] (1) Dissolve 36 mg (0.1 mmol) gentiopicroside in 1.0 mL ddH2O to obtain a clear and transparent solution A, and dissolve 95.1 mg (0.6 mmol) strontium chloride in 1.0 mL ddH2O to obtain a clear and transparent solution B;

[0065] (2) Stir continuously at a speed of 500 rpm / min, add solution A to solution B at a dropping rate of 60 drops / min, then add 0.5 mL ddH2O, and stir continuously for 2 hours to completely transfer the solution, to obtain mixed solution C;

[0066] (3) Add 10 mL of anhydrous ethanol to the mixture C, heat to 50 °C under argon protection and light protection, and stir for 24 hours to obtain solution D;

[0067] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0068] (5) The viscous liquid E was freeze-dried at -40℃ and 800Pa vacuum for 48 hours, and then recrystallized 4 times with methanol to obtain a white powder, which is the strontium gentiopicroside complex.

[0069] Example 3: A method for preparing a gentiopicroside strontium complex, comprising the following steps:

[0070] (1) Dissolve 71 mg (0.2 mmol) gentiopicroside in 1.0 mL ddH2O to obtain a clear and transparent solution A, and dissolve 63.4 mg (0.4 mmol) strontium chloride in 1.0 mL ddH2O to obtain a clear and transparent solution B;

[0071] (2) While stirring continuously at 800 rpm / min, add solution A to solution B at a dropping rate of 120 drops / min, then add 0.8 mL ddH2O, and continue stirring for 4 hours to completely transfer the solution, thus obtaining mixed solution C.

[0072] (3) Add 30 mL of anhydrous ethanol to the mixture C, heat to 40 °C under argon protection and light protection, and stir for 36 hours to obtain solution D;

[0073] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0074] (5) The viscous liquid E was freeze-dried at -60℃ and 900Pa vacuum for 40 hours, and then recrystallized three times with methanol to obtain a white powder, which is the gentiopicrin strontium complex.

[0075] Example 4: A method for preparing a gentiopicroside strontium complex, comprising the following steps:

[0076] (1) Dissolve 71.3 mg (0.2 mmol) gentiopicroside in 1.0 mL ddH2O to obtain a clear and transparent solution A, and dissolve 95.1 mg (0.6 mmol) strontium chloride in 1.0 mL ddH2O to obtain a clear and transparent solution B;

[0077] (2) Stir continuously at a speed of 700 rpm / min, add solution A to solution B at a dropping rate of 70 drops / min, then add 1 mL ddH2O, and continue stirring for 2-6 hours to completely transfer the solution and obtain mixed solution C.

[0078] (3) Add 10 mL of anhydrous ethanol to the mixture C, heat to 40 °C under argon protection and light protection, and stir for 48 hours to obtain solution D;

[0079] (4) Cool solution D to room temperature and remove ethanol by rotary evaporation at no more than 40°C, leaving a viscous liquid E;

[0080] (5) The viscous liquid E was freeze-dried at -50℃ and 1000Pa vacuum for 30 hours, and then recrystallized twice with methanol to obtain a white powder, which is the gentiopicrin strontium complex.

[0081] The gentiopicroside-strontium complex prepared by this invention possesses the characteristics of both gentiopicroside and strontium. On the one hand, it enables the effective release of strontium in vivo, allowing it to exert its effects on reducing inflammation and promoting osteoblast proliferation, thereby altering the immune environment of bone and promoting the proliferation and differentiation of bone repair-related cells. On the other hand, it improves the stability and bioavailability of gentiopicroside monomer.

Claims

1. A method for preparing a gentiopicroside strontium complex, characterized in that, Includes the following steps: (1) Dissolve 0.1-0.3 mmol of gentiopicroside in 1.0 mL of ddH2O to obtain a clear and transparent solution A. Dissolve strontium chloride in 1.0 mL of ddH2O at a molar ratio of 1:2-6 to obtain a clear and transparent solution B. (2) Under continuous stirring, solution A is added to solution B at a dropping rate of 60-120 drops / min. After the addition is completed, a mixed solution C is obtained. (3) Add anhydrous ethanol to the mixture C, heat to 40-60℃ under argon protection and light protection, and mix thoroughly to obtain solution D; (4) Cool solution D to room temperature, remove ethanol by rotary evaporation at a temperature not exceeding 40°C, and obtain a viscous liquid E; (5) Freeze-dry the viscous liquid E and recrystallize it with methanol to obtain a white powder, which is the gentiopicroside strontium complex.

2. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, The stirring speed in step (2) is 500-800 rpm / min, and the stirring time is 2-6 hours.

3. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, After the addition in step (2) is completed, add 0.5-1.0 mL of ddH2O to ensure complete transfer.

4. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, Add 10-30 mL of anhydrous ethanol in step (3).

5. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, In step (3), the mixture is thoroughly mixed by stirring for 24-48 hours.

6. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, The freeze-drying in step (5) is carried out continuously for 24-48 hours at a vacuum of -80 to -40°C and 800-1000 Pa.

7. The method for preparing the gentiopicroside strontium complex according to claim 1, characterized in that, The recrystallization process in step (5) is repeated 2-4 times.

8. The gentiopicrin strontium complex prepared by the method according to any one of claims 1-7, characterized in that: Strontium ions are complexed on the hydroxyl and carbonyl groups of gentiopicroside, accounting for 20-40% of the complex, and the particle size of the complex is smaller than that of the gentiopicroside monomer.

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