Glycyrrhizic acid-artesunate supramolecule as well as preparation method and application thereof
By preparing glycyrrhizic acid-artesunate supramolecules, the problems of low water solubility and oral utilization of artesunate were solved, the intestinal targeting and anti-inflammatory effects were improved, and a more convenient clinical application solution was provided.
Patent Information
- Application Number
- CN202510841360.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-23
- Publication Date
- 2025-09-26
AI Technical Summary
The clinical application of artesunate (ARS) is limited by its low water solubility, nonspecific distribution, low oral availability and poor anti-inflammatory effect.
Glycyrrhizic acid-artesunate supramolecules were prepared by a two-phase interface fusion method. The amphiphilic properties of glycyrrhizic acid were utilized to combine with artesunate to form a stable nanostructure, thereby enhancing its water solubility and intestinal targeting.
It improves the water solubility and oral availability of artesunate, enhances the anti-inflammatory effect, significantly improves intestinal inflammation, and provides a more convenient clinical dosing regimen.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to a supramolecular drug and a preparation method and application thereof, in particular to a glycyrrhizic acid-artesunate supramolecular drug and a preparation method and application thereof. Background Art
[0002] Artemisinin (ARS), a derivative of artemisinin, the main active ingredient in the traditional Chinese medicine Artemisia annua (Artemisia annua), is approved by the FDA as a first-line treatment for cerebral malaria and various forms of severe malaria. In addition to its antimalarial effects, ARS also possesses significant anti-inflammatory and immunomodulatory properties. However, ARS's low water solubility and nonspecific distribution result in low oral availability, rapid degradation and elimination, a lack of targeting, and poor anti-inflammatory efficacy, limiting its clinical application.
[0003] Glycyrrhizic acid (GA), a triterpenoid saponin extracted from the root of licorice, exhibits multiple biological activities, including anti-inflammatory, antiviral, and immunomodulatory activities. GA possesses amphiphilic properties due to its hydrophobic triterpenoid skeleton and hydrophilic glucuronic acid structure, enabling it to self-assemble into various supramolecular structures, such as micelles and hydrogels. However, no studies have yet demonstrated the binding of GA to ARS. Summary of the Invention
[0004] Objectives of the invention: The first objective of the present invention is to provide a glycyrrhizic acid-artesunate supramolecule with intestinal targeting, good ARS water solubility, and high oral utilization. The second objective of the present invention is to provide a method for preparing the glycyrrhizic acid / artesunate supramolecule. The third objective of the present invention is to provide the use of the glycyrrhizic acid / artesunate supramolecule in the preparation of anti-inflammatory drugs.
[0005] Technical solution: The method for preparing a glycyrrhizic acid-artesunate supramolecule (AG) according to the present invention comprises the following steps:
[0006] (1) dissolving ARS in an organic solvent to obtain an organic phase ARS solution;
[0007] (2) dissolving GA in deionized water to obtain an aqueous GA solution;
[0008] (3) adding the organic phase to the aqueous phase and stirring to volatilize the organic solvent;
[0009] (4) The solution after the organic solvent is evaporated is sonicated to obtain AG.
[0010] Furthermore, in step (1), the organic solvent is acetone or chloroform, and the concentration of the artesunate solution is 5-10 mg / mL. In step (2), the concentration of the GA solution is 1-4 mg / mL. The method for preparing the aqueous phase is water bath heating, the water bath heating temperature is 80-85°C, and the water bath heating time is 10-15 minutes. In step (3), the volume ratio of the aqueous phase to the organic phase is (5-10):1, and the organic phase is slowly added dropwise to the aqueous phase using a syringe at a uniform speed, the stirring speed is 500-600 rpm, and the stirring time is 6-8 hours. In step (4), the ultrasonic power is 67-90W, and the ultrasonic time is 10-15 minutes.
[0011] The present invention also includes the glycyrrhizic acid-artesunate supramolecule obtained by the preparation method.
[0012] The present invention also includes the use of the glycyrrhizic acid / artesunate supramolecule in the preparation of anti-inflammatory drugs.
[0013] Furthermore, the inflammation is intestinal inflammation.
[0014] The present invention addresses the problems of ARS (ARS) such as poor water solubility, nonspecific distribution, and low oral availability. By successfully preparing glycyrrhizic acid-artesunate supramolecules through a two-phase interfacial fusion method, the glycyrrhizic acid / artesunate supramolecules not only improve the inherent problems of ARS drugs but also exert a synergistic effect, enhancing the drug's anti-inflammatory effects. Using BALB / c male mice as test animals, the present invention determined the inhibitory effect of the glycyrrhizic acid-artesunate supramolecule on inflammatory responses by evaluating the survival rate, body weight, DAI, organ pathology, and inflammatory damage of the test mice after LPS-induced inflammatory injury. The glycyrrhizic acid-artesunate supramolecules significantly improved weight loss and duodenal inflammatory damage in mice with LPS-induced enteritis, providing a reference for the prevention and treatment of inflammatory diseases in animals.
[0015] Beneficial effects: Compared with the prior art, the present invention has the following advantages:
[0016] (1) This invention combines GA and ARS for the first time to form a glycyrrhizic acid-artesunate supramolecule (AG). The resulting AG has a long shelf life and good stability. The preparation of AG does not require traditional chemical carrier materials, thus reducing drug costs.
[0017] (2) The AG prepared by the present invention improves the water solubility of ARS and has intestinal targeting, solving the shortcomings of ARS such as poor water solubility and poor oral utilization, and can enhance the anti-inflammatory effect of ARS, making oral administration more convenient and facilitating clinical administration, providing new ideas for the development and application of supramolecular complexes of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1The results of water solubility of ARS, GA and AG in Example 2 are shown;
[0019] Figure 2 The ARS, GA, and AG scanning electron microscopy results in Example 2 are shown;
[0020] Figure 3 These are the infrared results of ARS, GA, ARS+GA, and AG in Example 2;
[0021] Figure 4 The XRD results of ARS, GA and AG in Example 2 are shown;
[0022] Figure 5 The graph shows the test results of the size and zeta potential of AG stored at 4°C and 37°C for 28 days in Example 2;
[0023] Figure 6 This is a graph showing the effect of AG on the body weight and DAI of mice with LPS-induced inflammation in Example 2;
[0024] Figure 7 Comparison of HE sections of spleens of the experimental mice in each group in Example 2;
[0025] Figure 8 Comparison of HE sections of the liver of each group of experimental mice in Example 2;
[0026] Figure 9 Comparison of HE sections of the duodenum of each group of experimental mice in Example 2;
[0027] Figure 10 This is a graph showing the RT-qPCR results of the duodenum of each group of experimental mice in Example 2. DETAILED DESCRIPTION
[0028] The technical solution of the present invention will be further described below with reference to the accompanying drawings.
[0029] Example 1 Optimization of preparation conditions
[0030] (1) Organic phase / aqueous phase synergistic dissolution:
[0031] First, four 5mL EP tubes were filled with 5mg of ARS powder and 1mL of methanol, 5mg of ARS powder and 1mL of ethanol, and 5mg of ARS powder and 1mL of acetone, respectively. After mixing thoroughly and letting the mixture stand for 30 minutes, the optimal organic solvent for ARS was screened. The solubility of ARS in equal volumes of methanol, ethanol, and acetone under fixed conditions was compared. The results showed that ARS had better solubility in acetone, and acetone was ultimately used as the organic solvent. By slowly adding the organic phase to the aqueous phase and evaporating the acetone, the two-phase system was gradually merged, avoiding drug precipitation or aggregation caused by direct mixing and improving compounding efficiency.
[0032] (2) Dynamic self-assembly process design:
[0033] To screen for the appropriate rotation speed and evaporation time, we compared the effects of magnetic stirring speeds of 600 rpm, 500 rpm, and 700 rpm on the self-assembly of ARS and GA, while keeping other conditions constant. First, three 20 mL beakers were added with 10 mL of a 1 mg / mL GA solution and 1 mL of a 5 mg ARS solution dissolved in acetone. The mixture was placed on magnetic stirrers at 600 rpm, 500 rpm, and 700 rpm, respectively, and stirred in a fume hood for 2 hours. It was found that drug self-assembly was more uniform at 600 rpm. Through multiple experimental screening, we found that at 600 rpm, acetone was completely evaporated within 6 hours without losing any water. The 600 rpm magnetic stirring provides moderate shear force, promoting contact between the two phases. Simultaneously, the acetone was slowly evaporated within the fume hood (6 hours), allowing the ARS and GA molecules to gradually self-assemble through non-covalent bonds (such as hydrogen bonds and hydrophobic interactions). Different from the conventional simple mixing or freeze-drying method, this method achieves more uniform supramolecular formation through the synergistic effect of dynamic stirring and solvent evaporation to enhance drug loading rate and stability.
[0034] (3) Ultrasonic-assisted nano-processing:
[0035] In order to determine the optimal ultrasonic-assisted processing power parameters, we set up three groups of ultrasonic powers of 30W, 67W, and 100W for comparative experiments. First, three 15ml EP tubes were taken, and 10mL of ARS / GA self-assembly solution was added to each tube, which was rotated at 600rpm for 6h. Then the three EP tubes were placed in ultrasonic instruments with 30W, 67W, and 100W powers for 10 minutes. By comparison, we found that the higher the ultrasonic power, the stronger the cavitation effect, but the risk of overheating also increased. Finally, 67W power ultrasound for 10 minutes was adopted as the ultrasonic-assisted processing condition. The ultrasonic step is not only used for homogenization, but it may also adjust the supramolecular particle size through the cavitation effect to form a more stable nanostructure, which is crucial for the solubility of the drug. Ultrasound at 67W power for 10 minutes can further nanosize the supramolecular particles and improve dispersibility and bioavailability.
[0036] Example 2
[0037] (1) Dissolve 10 mg of ARS in 1 mL of acetone and stir to obtain a uniform organic phase ARS solution.
[0038] (2) Add 10 mg of GA to 10 mL of ultrapure water and heat in a water bath at 80°C for 10 min to obtain an aqueous GA solution;
[0039] (3) The aqueous GA solution was added to a 20 mL beaker and placed on a magnetic stirrer with the speed adjusted to 600 rpm. The organic ARS solution was then extracted using a syringe and slowly and uniformly added (at a rate of 3 seconds per drop) to the stirring aqueous GA solution. The mixture was stirred in a fume hood for 6 h until the acetone was completely evaporated.
[0040] (4) The solution from which the acetone has evaporated is placed in an ultrasonicator and ultrasonicated for 10 minutes at a power of 67 W to obtain the glycyrrhizic acid-artesunate supramolecule (AG).
[0041] Characterization assays:
[0042] (1) Analysis of water solubility results
[0043] Take 4 5mL EP tubes, add 2mg ARS powder and 4mL ultrapure water, 4mg GA powder and 4mL ultrapure water, 2mg ARS + 4mg GA powder and 4mL ultrapure water and 4mL AG of this embodiment into the tubes respectively, mix well and let it stand for 30 minutes to observe the dissolution. ARS is a fat-soluble drug, is hydrophobic, easily soluble in organic solvents (such as acetone, methanol, DMSO), and is difficult to dissolve in water. GA is an amphiphilic molecule containing hydrophilic glycoside chains and hydrophobic triterpene structures, and the sugar chains give it a certain water solubility. However, at room temperature, the solubility of GA in pure water is about 1-2mg / mL, which is slightly soluble. The solubility can only be increased by heating the water bath to 80°C, but GA will still precipitate after cooling. ARS, GA and AG were tested for water solubility, and the results are as follows. Figure 1 It can be clearly seen that ARS and GA are almost insoluble in water, while AG is completely soluble in water without any precipitation or precipitation.
[0044] (2) Analysis of scanning electron microscopy results
[0045] Scanning electron microscopy was performed on ARS, GA and AG in this embodiment, and the results were as follows: Figure 2 shown. Figure 2 This is the transmission electron microscopy analysis result of AG; it can be clearly found that after ARS and GA are self-assembled, their morphology undergoes a significant and clear change to form a glycyrrhizic acid-artesunate supramolecule.
[0046] (3) Analysis of Fourier transform infrared spectroscopy results
[0047] In this example, ARS, GA, AG and ARS+GA (ARS and GA were directly physically mixed at a mass ratio of 1:2) were mixed with KBr powder and ground into tablets. -1 After infrared scanning within the wavelength range, the infrared spectrum results are shown in Figure 3 .Depend on Figure 3It can be seen that by comparing the infrared spectra of ARS+GA, ARS and GA, no new characteristic peaks appear in the spectrum of the physical mixture of ARS and GA. This shows that there is no chemical reaction in the formation process of ARS and GA, and it is a simple physical combination. -1 The broad peak formed shows a decrease in the molecular association degree compared with the large peak of the raw material. -1 The weak peaks on the left and right confirm that the enhanced symmetry of the molecules after binding leads to weak peaks. Compared with the raw material, the degree of molecular association between AG molecules is reduced, and the main functional groups are retained. These results indicate that AG was successfully synthesized and has certain differences from its raw material, not just a simple physical bond.
[0048] (4) Analysis of X-ray diffraction spectrum results
[0049] X-ray diffraction analysis was performed on ARS, GA and AG in this embodiment. The results are as follows: Figure 4 As shown. Figure 4 It can be seen that the XRD results of ARS show that it is crystalline, while the XRD results of GA and AG show that they are amorphous. The above results indicate that AG is not a simple mechanical mixture of ARS and GA, but a complex amorphous combination.
[0050] (5) Particle size and potential measurement
[0051] The particle size and potential of the AG obtained in this example were measured, and the results are shown in Table 1.
[0052] Table 1 Particle size, PDI and Zeta potential of AG (n=3)
[0053] sample AG Particle size (nm) 109.97±10.58 PDI 0.23±0.03 Zeta potential (mV) -25.65±9.04
[0054] As can be seen from Table 1, the average particle size of the AG supramolecule is 109.97 ± 10.58 nm, the Zeta potential is -25.65 ± 9.04 mV, and the polydispersity index (PDI) is 0.23 ± 0.03. The results show that the prepared AG has a small particle size and a narrow particle size distribution.
[0055] (6) Stability analysis
[0056] The AG obtained in this example was stored at 4°C and 37°C for different time periods and subjected to Malvern analysis. Figure 5 shown.
[0057] The particle size and potential of AG stored at 4℃ and 37℃ for 28 days were measured on days 0, 1, 3, 5, 7, 14, 21 and 28 respectively. The results are as follows: Figure 5 shown. Figure 5The test results of the size and zeta potential of AG stored at 4°C and 37°C for 28 days in Example 2 are shown in Figure 2; wherein, Figure A is the size result of AG stored at 4°C for 28 days, Figure B is the zeta potential result of AG stored at 4°C for 28 days, Figure C is the size result of AG stored at 37°C for 28 days, and Figure D is the zeta potential result of AG stored at 37°C for 28 days. Figure 5 As can be seen from the results, the particle size and potential of AG stored at 4°C and 37°C remained largely unchanged over the 28-day test period. The overall particle size and potential changes of AG stored at 4°C were smaller than those at 37°C. This suggests that the prepared AG is more stable and easier to store at 4°C.
[0058] (7) Performance testing
[0059] 1) Effects of AG on body weight and DAI in mice with LPS-induced enteritis
[0060] Specific test process:
[0061] Forty-two BALB / c male mice (weighing 20±2g) were randomly divided into the Con group, LPS, LPS+ARS, LPS+GA, LPS+MDCM (ARS and GA were physically mixed at a mass ratio of 1:2), LPS+AG, and LPS+dexamethasone (DEX) groups, with six mice per group. The inflammatory model was induced by intraperitoneal injection of 3mg / kg LPS. Within 7 days of modeling, mice in the LPS+AG group were gavaged with AG (20mg / kg); mice in the LPS+ARS group were gavaged with ARS (20mg / kg per mouse, based on the AG concentration); mice in the LPS+GA group were gavaged with GA (20mg / kg); mice in the LPS+MDCM group were gavaged with ARS+GA (20mg / kg); and mice in the LPS+DEX group were gavaged with DEX (1mg / kg). Mice in the Con group were gavaged with an equal volume of normal saline, while mice in the LPS group were left untreated. This was repeated once daily for 7 consecutive days. To ensure that each group of mice received the same stimulus, all mice received the same treatment. During the experiment, the mice were monitored daily for weight, stool consistency, occult blood or bleeding, and clinical symptoms. After seven days of feeding, the mice were anesthetized and sacrificed, and their liver, spleen, duodenum, and other tissues were collected for testing.
[0062] The weight, feces consistency, occult blood and bleeding of mice were recorded and sorted every day during the experiment. Figure 6 shown. Figure 6The results of the effect of AG on the weight and DAI of mice with LPS-induced inflammation in Example 2 are shown in Figure A, which shows the weight changes of mice within 7 days, B shows the weight comparison of mice on the seventh day, C shows the DAI changes of mice within 7 days, and D shows the DAI comparison of mice on the seventh day. Figure 6 It can be seen that the AG in the GA prepared in this example significantly improved the body weight and feces of mice with LPS-induced enteritis compared with other groups, effectively inhibited the weight loss caused by LPS, and reduced the DAI score.
[0063] 2) Effects of AG on the liver and spleen of mice with LPS-induced enteritis
[0064] The liver and spleen of the experimental mice were collected and HE staining was performed to evaluate the pathological changes in organ tissues. Figure 7 、 Figure 8 shown. Figure 7 This is a comparison of HE sections of the spleen of each group of experimental mice in Example 2. Figure 7 In the middle, the pink arrow indicates hemorrhage, the yellow arrow indicates lymphoproliferation, and the green arrow indicates spleen atrophy. Figure 8 This is a comparison of HE sections of the liver of each group of experimental mice in Example 2. Figure 8 In the figure, yellow arrows indicate liver hemorrhage and congestion, blue arrows indicate neutrophil infiltration, and black arrows indicate lymphocyte infiltration. Figure 7-8 As can be seen, the liver and spleen of mice in the Con group showed no obvious pathological changes, while the liver and spleen of the LPS group showed damage such as hemorrhage and inflammatory cell infiltration. Compared with the other groups, oral administration of AG greatly alleviated the pathological damage of LPS to the spleen and liver.
[0065] 3) Effects of AG on the duodenum of mice with LPS-induced enteritis
[0066] The duodenum of the experimental mice was collected for HE staining and RT-qPCR to evaluate the therapeutic effect of AG on mouse enteritis. Figure 9 、 Figure 10 shown. Figure 9 This is a comparison of HE sections of the duodenum of each group of experimental mice in Example 2. Figure 9 In the figure, red arrows indicate inflammatory cell infiltration, green arrows indicate goblet cell reduction and crypt destruction, and pink arrows indicate hemorrhage. Figure 9 The results showed that no obvious pathological changes were observed in the duodenum of mice in the Con group, while the duodenum of mice in the LPS group showed damage to epithelial cells, reduced goblet cells, and crypt destruction, accompanied by inflammatory cell infiltration. Compared with the other treatment groups, oral administration of AG significantly alleviated the inflammatory damage of LPS to the duodenal mucosa. Figure 10The following are the RT-qPCR results of the duodenum of each group of experimental mice in Example 2, wherein A is the relative expression level of the inflammatory factor interleukin-1β gene in the duodenum of each group of mice, and B is the relative expression level of the inflammatory factor interleukin-6 gene in the duodenum of each group of mice. Figure 10 As shown, LPS significantly increased the mRNA expression levels of IL-1β and IL-6 in the duodenum of mice compared to the Con group. After oral administration of AG, the expression of IL-1β and IL-6 was significantly reduced compared to the LPS group. These results demonstrate that the AG supramolecules prepared in this invention can significantly inhibit the inflammatory response induced by LPS and effectively alleviate pathological changes in the duodenum of mice.
Claims
1. A method for preparing a glycyrrhizic acid-artesunate supramolecule, characterized in that: The steps include: (1) dissolving artesunate in an organic solvent to obtain an organic phase artesunate solution; (2) dissolving glycyrrhizic acid in deionized water to obtain an aqueous glycyrrhizic acid solution; (3) adding the organic phase to the aqueous phase and stirring to volatilize the organic solvent; (4) After the organic solvent is evaporated, the solution is sonicated to obtain glycyrrhizic acid-artesunate supramolecules.
2. The preparation method according to claim 1, characterized in that In step (1), the organic solvent is acetone or chloroform.
3. The preparation method according to claim 1, characterized in that In step (1), the concentration of the artesunate solution is 5-10 mg / mL.
4. The preparation method according to claim 1, characterized in that In step (2), the concentration of the glycyrrhizic acid solution is 1-4 mg / mL.
5. The preparation method according to claim 1, characterized in that In step (2), the method for preparing the aqueous phase is heating in a water bath, the water bath heating temperature is 80-85° C., and the water bath heating time is 10-15 min.
6. The preparation method according to claim 1, characterized in that In step (3), the volume ratio of the aqueous phase to the organic phase is (5-10):1, and the organic phase is slowly added dropwise to the aqueous phase using a syringe at a uniform speed, with a stirring speed of 500-600 rpm and a stirring time of 6-8 h.
7. The preparation method according to claim 1, characterized in that In step (4), the ultrasonic power is 67-90W, and the ultrasonic time is 10-15 minutes.
8. The glycyrrhizic acid-artesunate supramolecule obtained by the preparation method according to any one of claims 1 to 7.
9. Use of the glycyrrhizic acid-artesunate supramolecule according to claim 8 in the preparation of anti-inflammatory drugs.
10. The use according to claim 9, characterized in that The inflammation is intestinal inflammation.