Ternary eutectic solvent as well as preparation method and application thereof

Abstract

The invention relates to the technical field of supramolecules, in particular to a ternary eutectic solvent and a preparation method and application thereof.The ternary eutectic solvent is prepared from a component A, a component B and a component C. The component A is a hydrogen bond donor, the component B is a hydrogen bond receptor, and the component C is adenosine. The intermolecular interaction mode of the ternary eutectic solvent breaks the original strong acting force between adenosine molecules, so that adenosine can be uniformly dispersed in a ternary DES system in the form of a single molecule or a small aggregate, and the solubility of adenosine is remarkably improved. Meanwhile, due to the existence of the ternary DES, the dissolution and release behaviors of the medicine in the body are changed, the absorption of the medicine is promoted, and the bioavailability of adenosine is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of supramolecular technology, and in particular to a ternary deep eutectic solvent and a preparation method and application thereof. Background Art

[0002] Adenosine is an endogenous nucleoside found throughout human cells and possesses a wide range of physiological functions within the body. For example, adenosine regulates the cardiovascular system, with effects such as vasodilation, blood pressure lowering, and anti-arrhythmic properties. In the nervous system, adenosine participates in sleep regulation and neuroprotection, and also plays an important regulatory role in the immune system. However, adenosine has a low solubility in water, which greatly limits its application in pharmaceutical formulations and its bioavailability. This low solubility of adenosine results in slow and incomplete dissolution and absorption of the drug within the body, making it difficult for the drug to effectively reach its target site, thereby affecting its efficacy.

[0003] Currently, common methods for improving drug solubility and bioavailability include salt formation, the use of surfactants, cyclodextrin inclusion technology, and nanoformulation technology. However, these methods have many limitations. Salt formation may alter the chemical properties and stability of the drug, and not all drugs can be effectively improved in solubility through salt formation. The use of surfactants may cause toxic side effects and has limited solubilization effects. Cyclodextrin inclusion technology has strict requirements on the type and ratio of cyclodextrin, is costly, and has an unstable inclusion rate. Although nanoformulation technology can significantly improve drug solubility and bioavailability, its preparation process is complex, requires specialized equipment and techniques, and is costly, making it unsuitable for large-scale production and clinical application.

[0004] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a ternary deep eutectic solvent and a preparation method and application thereof, so as to solve the problem of low solubility of adenosine in water.

[0006] The technical solutions adopted by the present invention to solve the above technical problems are as follows:

[0007] In a first aspect of the present invention, a ternary deep eutectic solvent is provided, wherein the ternary deep eutectic solvent comprises component A, component B and component C, wherein component A is a hydrogen bond donor, component B is a hydrogen bond acceptor, and component C is adenosine.

[0008] Preferably, the component A is selected from one or more of malic acid, tartaric acid, citric acid and salicylic acid.

[0009] Preferably, the component B is selected from one or both of L-carnitine and betaine.

[0010] Preferably, in the ternary deep eutectic solvent, the molar ratio of component A, component B and component C is (5-15):(5-15):1.

[0011] Preferably, in the ternary deep eutectic solvent, the molar ratio of component A, component B and component C is (8-12):(8-12):1.

[0012] A second aspect of the present invention provides a method for preparing the above-mentioned ternary deep eutectic solvent, the preparation method comprising the following steps:

[0013] Mixing component A and component B, and performing a first heating and stirring process to obtain a mixed solution;

[0014] Component C is added to the mixed solution, and a second heating and stirring treatment is performed to obtain the ternary deep eutectic solvent.

[0015] Preferably, the temperature of the first heating and stirring treatment is 50-80° C., and the time is 1-3 hours.

[0016] Preferably, the temperature of the second heating and stirring treatment is 35-90° C., and the time is 3-6 hours.

[0017] A third aspect of the present invention provides use of the above-mentioned ternary deep eutectic solvent or the ternary deep eutectic solvent prepared by the above-mentioned preparation method in the preparation of drugs.

[0018] Beneficial effects:

[0019] The present invention discloses a ternary low eutectic solvent and its preparation method and application. The ternary low eutectic solvent provided by the present invention has the following advantages: (1) Significantly improve the solubility of adenosine: The ternary low eutectic solvent provided by the present invention can increase the solubility of adenosine by about 100 times, which has extremely significant advantages compared with traditional solvent systems or solubilization methods. The high solubility allows the concentration of adenosine in pharmaceutical preparations to be greatly increased, providing the possibility for the development of efficient pharmaceutical dosage forms. (2) Improved bioavailability: The significant increase in solubility directly promotes the dissolution and absorption process of adenosine in the body, allowing the drug to reach the target site faster and more effectively, greatly improving the bioavailability of adenosine. Experiments show that after using the ternary low eutectic solvent of the present invention, the peak blood concentration of adenosine in animals is significantly increased, the duration of drug action is prolonged, and the efficacy of the drug is significantly improved. (3) Simple preparation process: The preparation method of the ternary low eutectic solvent provided by the present invention can be completed by simple heating and stirring, without the need for complex equipment and technology, and is simple to operate, easy to mass produce, reducing production costs, and conducive to industrial promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the adenosine molecular structure of the present invention.

[0021] Figure 2 The ternary DES NMR spectrum of malic acid betaine adenosine obtained in Experimental Example 2 of the present invention is ( 1 H-NMR) chart.

[0022] Figure 3 This is a comparison chart of infrared spectra of malic acid, betaine, adenosine, malate betaine binary DES, and malate betaine adenosine ternary DES in Experimental Example 2 of the present invention.

[0023] Figure 4 Infrared spectra of the malic acid betaine adenosine ternary DES prepared in Example 2 of the present invention diluted with water in different proportions.

[0024] Figure 5 This is the negative control group containing only complete culture medium in the live and dead cell staining experiment in Experimental Example 2 of the present invention.

[0025] Figure 6 This is the experimental group of the malate betaine adenosine ternary DES sample with a concentration of 2 mg / ml in the live and dead cell staining experiment in Experimental Example 2 of the present invention.

[0026] Figure 7 This is the optimized structure diagram of the malate betaine adenosine ternary DES of Experimental Example 2 of the present invention.

[0027] Figure 8 The ternary DES NMR spectrum of tartrate betaine adenosine obtained in Experimental Example 4 of the present invention is ( 1 H-NMR) chart.

[0028] Figure 9 This is a comparison chart of infrared spectra of tartaric acid, betaine, adenosine, tartrate betaine binary DES, and tartrate betaine adenosine ternary DES in Experimental Example 4 of the present invention.

[0029] Figure 10 Infrared spectra of tartrate betaine adenosine ternary DES of Example 4 of the present invention diluted with water in different proportions.

[0030] Figure 11 This is the optimized structure diagram of the ternary DES of betaine tartrate adenosine of Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0031] The present invention provides a ternary deep eutectic solvent and its preparation method and application. To make the objectives, technical solutions, and effects of the present invention more clear and explicit, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0032] As a new type of green solvent, deep eutectic solvent (DES) has received widespread attention in the field of pharmaceutical formulations in recent years. DES is usually formed by hydrogen bond acceptors (quaternary ammonium salts) and hydrogen bond donors (such as organic acids, polyols, amides, etc.) through hydrogen bond interactions. It has the advantages of low volatility, high stability, and biodegradability. However, traditional binary DES still has shortcomings in improving the solubility of certain drugs. For poorly soluble drugs such as adenosine, a single binary DES is difficult to achieve a significant increase in its solubility. Therefore, the development of a new and efficient method and composition for improving the solubility and bioavailability of adenosine has important practical significance and clinical application value.

[0033] Furthermore, compared to solid cocrystals, DES can significantly improve drug solubility. Many solid drugs have low solubility in water or other common solvents. However, the unique intermolecular interactions and structural characteristics of DES enable them to dissolve a wide range of drugs, evenly dispersing them in a molecular state throughout the system, which helps improve drug bioavailability. DES generally exhibits good chemical and thermal stability. At room temperature and under standard storage conditions, the drug contained in DES maintains a stable chemical structure and properties, making it less susceptible to degradation, deterioration, or crystal transformation, facilitating long-term storage and quality control. By varying the composition and formulation of DES, its physicochemical properties, such as viscosity, density, and surface tension, can be flexibly adjusted to suit diverse drug delivery requirements. For example, for topical drug delivery, the viscosity of DES can be adjusted to facilitate application and adhesion to the administration site. DES enables stable storage and controlled release of drugs while minimizing toxic side effects on normal tissues.

[0034] Based on this, an embodiment of the present invention provides a ternary deep eutectic solvent, which is prepared by including component A, component B and component C, wherein component A is a hydrogen bond donor, component B is a hydrogen bond acceptor, and component C is adenosine.

[0035] In the ternary DES of the embodiment of the present invention, the hydrogen bond acceptor has good hydrophilicity and cationic properties, and the carboxyl and hydroxyl groups of the hydrogen bond donor can form strong hydrogen bond interactions with the hydrogen bond acceptor to construct the framework structure of the binary DES. The active groups such as hydroxyl and amino groups in the adenosine molecule (its structural diagram is shown in FIG. Figure 1The ternary DES (shown in Figure 2) can further interact with hydrogen bond acceptors and hydrogen bond donors through hydrogen bonds and van der Waals forces, forming a stable ternary DES system. This unique intermolecular interaction mode breaks the inherent strong forces between adenosine molecules, allowing them to be evenly dispersed in the ternary DES system as single molecules or small aggregates, significantly improving adenosine solubility. Furthermore, the presence of the ternary DES alters the drug's dissolution and release behavior in the body, promoting drug absorption and significantly enhancing adenosine bioavailability.

[0036] In some embodiments, the component A is selected from one or more of malic acid, tartaric acid, citric acid, and salicylic acid.

[0037] In some embodiments, the component B is selected from one or both of L-carnitine and betaine.

[0038] The raw materials malic acid, tartaric acid, citric acid, salicylic acid, L-carnitine, and betaine used in the embodiments of the present invention are all naturally occurring and biodegradable substances. No toxic or harmful organic solvents are required in the preparation process, which is in line with the concept of green chemistry and is environmentally friendly.

[0039] In some embodiments, in the ternary deep eutectic solvent, the molar ratio of component A, component B, and component C is (5-15):(5-15):1.

[0040] This ratio can significantly lower the eutectic point of the mixed system (usually lower than the melting point of each individual component) through the synergistic effects of hydrogen bonds, ionic bonds, or van der Waals forces between the components, forming a stable eutectic phase. For example, when the ratio of component C is low, the appropriate amount of components A / B can serve as a "structural skeleton," forming a dense hydrogen bond network with component C, thereby minimizing the free energy of the system and achieving a lower melting temperature.

[0041] In some preferred embodiments, in the ternary deep eutectic solvent, the molar ratio of component A, component B and component C is (8-12):(8-12):1.

[0042] The present invention also provides a method for preparing the above-mentioned ternary deep eutectic solvent, which comprises the following steps:

[0043] Mixing component A and component B, and performing a first heating and stirring process to obtain a mixed solution;

[0044] Component C is added to the mixed solution, and a second heating and stirring treatment is performed to obtain the ternary deep eutectic solvent.

[0045] The preparation method of the embodiment of the present invention can be completed by simple heating and stirring, does not require complicated equipment and technology, is easy to operate, is easy to mass produce, reduces production costs, and is conducive to industrial promotion and application.

[0046] In some embodiments, the temperature of the first heating and stirring treatment is 50-80° C., and the time is 1-3 hours.

[0047] In some embodiments, the second heating and stirring treatment is performed at a temperature of 35-90° C. and for a time of 3-6 hours.

[0048] Setting the upper temperature limit can avoid thermal decomposition of most organic components, and controlling the lower time limit can ensure that the main reaction proceeds fully while avoiding side reactions caused by too long a time.

[0049] In some preferred embodiments, the temperature of the first heating and stirring treatment is 70° C., and the time is 1.5 hours.

[0050] In some preferred embodiments, the temperature of the second heating and stirring treatment is 80° C. and the time is 3 hours.

[0051] The embodiments of the present invention provide the use of the above-mentioned ternary deep eutectic solvent or the ternary deep eutectic solvent prepared by the above-mentioned preparation method in the preparation of drugs.

[0052] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them, and are intended only to illustrate the present invention and in no way limit the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are also within the scope of protection of the present invention.

[0053] Example 1

[0054] The preparation of malate L-carnitine adenosine ternary DES comprises the following steps:

[0055] Step 1: Accurately weigh 1 mol of malic acid (134 g / mol × 1 mol = 134 g) and 1 mol of L-carnitine (161.2 g / mol × 1 mol = 161.2 g), and add them to a three-necked flask equipped with a stirring device and a thermometer.

[0056] Step 2: Place the three-necked flask in a constant temperature water bath, heat to 60° C., and stir to react for 2 hours to obtain a uniform liquid, namely, L-carnitine malate binary DES.

[0057] Step 3: Add 0.33 mol of adenosine (267.25 g / mol×0.33 mol=88.19 g) to the above binary DES, and continue stirring and reacting at 60° C. for 4 hours to obtain a clear and transparent malate-L-carnitine adenosine ternary DES system.

[0058] Example 2

[0059] The preparation of malate betaine adenosine ternary DES comprises the following steps:

[0060] Step 1: Accurately weigh 1 mol of malic acid (134 g / mol × 1 mol = 134 g) and 1 mol of betaine (117.15 g / mol × 1 mol = 117.15 g), and add them into a three-necked flask equipped with a stirring device and a thermometer.

[0061] Step 2: Place the three-necked flask in a constant temperature water bath, heat to 60° C., and stir to react for 2 hours to obtain a uniform liquid, namely malic acid betaine dibasic DES.

[0062] Step 3: Add 0.33 mol of adenosine (267.25 g / mol×0.33 mol=88.19 g) to the above binary DES, and continue stirring and reacting at 60° C. for 4 hours to obtain a clear and transparent malic acid betaine adenosine ternary DES system.

[0063] The malic acid betaine binary DES and malic acid betaine adenosine ternary DES obtained in Example 2 were subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization, NMR spectrum such as Figure 2 shown. Figure 2 The H NMR spectrum shows that: through H NMR characterization, D2O was selected as the test solvent. From the H NMR spectrum, the two hydrogen atoms of adenosine on the N-containing five-membered ring and six-membered ring can be clearly found, and no obvious impurity peaks were observed. Infrared characterization was performed on malic acid, betaine, adenosine, and the obtained malic acid betaine dibasic DES and malic acid betaine adenosine ternary DES: The infrared spectrum is as follows Figure 3 As shown in the figure, the NH peak of adenosine shifted, indicating that the amino group of adenosine participated in the formation of hydrogen bonds. -1 and 1400-1800cm -1 The peak shape and position at 177° changed, indicating that adenosine and malic acid betaine formed a ternary DES through several interatomic hydrogen bonds.

[0064] In order to explore the dilution stability of the ternary deep eutectic solvent (DES) product prepared in Example 2, a series of gradient dilution strategies were used to accurately process the product. During the experiment, based on the preliminary experimental results and the properties of the product, a suitable dilution solvent was selected, and high-precision measuring instruments such as pipettes and volumetric flasks were used to prepare product solutions with different concentration gradients according to strict dilution ratios (such as mass ratio = 1:3, 1:4, 1:5, etc.). Subsequently, Fourier transform infrared spectroscopy was performed on the diluted products of different concentrations: the infrared spectrum is as follows: Figure 4 As shown, the position, intensity, and peak shape of infrared absorption peaks at different dilution ratios were analyzed to determine changes in intermolecular interactions. The infrared spectra of the diluted samples show that as the water content in DES increases, the intensity and width of the OH absorption peak at 3200-3600 nm increase, while the carbonyl absorption peak gradually disappears. Simultaneously, the intensity of the absorption peak in the infrared fingerprint region (1300-400 nm) decreases with the addition of water. This suggests that the addition of water gradually disrupts the hydrogen bond network in DES.

[0065] In order to investigate the cytotoxicity of the ternary deep eutectic solvent (DES) product prepared in Example 2, the live-dead cell staining method was used to conduct toxicity evaluation on L929 mouse fibroblasts. L929 cells were cultured at 5×10 4 The cells / well density was inoculated in a 24-well plate and pre-cultured in a 37°C, 5% CO2 incubator for 24 hours. The experimental group was added with a ternary DES sample solution with a final concentration of 2 mg / ml, and a negative control group containing only complete culture medium was set up. After continuing to culture for 24 hours, the culture medium in the wells was discarded, the cells were gently washed 3 times with PBS, and a staining working solution containing 2μM calcein and 4μM propidium iodide was added, and incubated at 37°C in the dark for 20 minutes. Cell images were observed and photographed using a fluorescence microscope, and 5 fields of view were randomly selected for analysis for each sample. Among them, Figure 5 The negative control group contained only complete culture medium. Figure 6 The experimental group was the ternary DES sample with a concentration of 2 mg / ml betaine malate adenosine. Figure 7 This is the optimized structure diagram of the malate betaine adenosine ternary DES.

[0066] The results showed that after staining, live cells in the experimental group showed bright green fluorescence, and the proportion of live cells was not significantly different from that in the negative control group (p>0.05). This indicates that the ternary DES sample at this concentration has no significant toxic effect on L929 cells and has good biocompatibility.

[0067] Example 3

[0068] The preparation of L-carnitine adenosine tartrate ternary DES comprises the following steps:

[0069] Step 1: Accurately weigh 1 mol of tartaric acid (150.09 g / mol × 1 mol = 150.09 g) and 1 mol of L-carnitine (161.2 g / mol × 1 mol = 161.2 g), and add them to a three-necked flask equipped with a stirring device and a thermometer.

[0070] Step 2: Place the three-necked flask in a constant temperature water bath, heat to 60° C., and stir to react for 2 hours to obtain a uniform liquid, namely, tartrate betaine dibasic DES.

[0071] Step 3: Add 0.33 mol of adenosine (267.25 g / mol×0.33 mol=88.19 g) to the above binary DES, and continue stirring and reacting at 60° C. for 4 hours to obtain a clear and transparent tartrate betaine adenosine ternary DES system.

[0072] Example 4

[0073] The preparation of ternary DES of betaine tartrate adenosine comprises the following steps:

[0074] Step 1: Accurately weigh 1 mol of tartaric acid (150.09 g / mol × 1 mol = 150.09 g) and 1 mol of betaine (117.15 g / mol × 1 mol = 117.15 g), and add them to a three-necked flask equipped with a stirring device and a thermometer.

[0075] Step 2: Place the three-necked flask in a constant temperature water bath, heat to 60° C., and stir to react for 2 hours to obtain a uniform liquid, namely, tartrate betaine dibasic DES.

[0076] Step 3: Add 0.33 mol of adenosine (267.25 g / mol×0.33 mol=88.19 g) to the above binary DES, and continue stirring and reacting at 60° C. for 4 hours to obtain a clear and transparent tartrate betaine adenosine ternary DES system.

[0077] The tartrate betaine binary DES and tartrate betaine adenosine ternary DES obtained in Example 4 were subjected to nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization, NMR spectrum such as Figure 8 shown. Figure 8 The nuclear magnetic resonance hydrogen spectrum shows that: through nuclear magnetic resonance hydrogen spectrum characterization, D2O is selected as the test solvent. From the nuclear magnetic hydrogen spectrum, the two hydrogen atoms of adenosine on the N-containing five-membered ring and six-membered ring can be clearly found, and no obvious impurity peaks are observed. Infrared characterization of tartaric acid, betaine, adenosine, and the obtained tartrate betaine dibasic DES and tartrate betaine adenosine ternary DES is carried out. The infrared spectrum is as follows Figure 9As shown in the figure, the NH peak of adenosine shifted, indicating that the amino group of adenosine participated in the formation of hydrogen bonds. -1 and 1400-1800cm -1 The peak shape and peak position at 177° changed, which proved that adenosine and tartrate betaine formed a ternary DES through several interatomic hydrogen bonds.

[0078] Figure 10 The following are infrared spectra of tartrate betaine adenosine ternary DES diluted with water at varying ratios. The FT-IR spectra of the diluted samples show that as the water content in the DES increases, the intensity and width of the OH absorption peak at 3200-3600 nm increase, while the carbonyl absorption peak gradually disappears. Simultaneously, the intensity of the absorption peak in the infrared fingerprint region (1300-400 nm) decreases with the addition of water. This suggests that the addition of water gradually disrupts the hydrogen bond network in the DES.

[0079] Figure 11 This is the optimized structure diagram of betaine adenosine tartrate ternary DES.

[0080] Adenosine solubilization effect test

[0081] 10 g of each of the ternary DES compositions prepared in Example 2 and Example 4 were placed in two 50 mL volumetric flasks. Deionized water was slowly added to the flasks with continuous stirring until the volume reached the volumetric flask mark, thereby preparing aqueous solutions containing the ternary DES.

[0082] Take another 50 mL volumetric flask, add 10 g of adenosine, then add deionized water and stir until the volume reaches the volumetric flask mark, which serves as the control group.

[0083] Place the three volumetric flasks in a 25°C constant temperature water bath shaker and shake for 24 hours to fully dissolve the adenosine. After shaking, remove the volumetric flasks and filter through a 0.45 μm microporous filter membrane. The filtrate is then used to determine the adenosine concentration using high performance liquid chromatography.

[0084] It was determined that the solubility of adenosine in the control group was 0.5 mg / mL, the solubility of adenosine in the malic acid betaine adenosine ternary DES system in Example 2 was 50 mg / mL, and the solubility of adenosine in the tartrate betaine adenosine ternary DES system in Example 4 was 48 mg / mL, which was about 100 times higher than that in the control group.

[0085] Test of the effect of surfactant on solubilization of adenosine

[0086] A 0.5% sodium dodecyl sulfate (SDS) aqueous solution was prepared, 10 g of adenosine was added to 50 mL of the SDS aqueous solution, and the solubility of adenosine therein was measured.

[0087] The solubility of adenosine in the system was measured to be 8 mg / mL, which is far lower than the solubility-enhancing effect of the ternary DES provided in the embodiment of the present invention on adenosine.

[0088] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A ternary deep eutectic solvent, characterized in that: The ternary deep eutectic solvent comprises component A, component B and component C, wherein component A is a hydrogen bond donor, component B is a hydrogen bond acceptor, and component C is adenosine.

2. The ternary deep eutectic solvent according to claim 1, characterized in that The component A is selected from one or more of malic acid, tartaric acid, citric acid and salicylic acid.

3. The ternary deep eutectic solvent according to claim 1, characterized in that The component B is selected from one or both of L-carnitine and betaine.

4. The ternary deep eutectic solvent according to claim 1, characterized in that In the ternary deep eutectic solvent, the molar ratio of component A, component B and component C is (5-15):(5-15):

1.

5. The ternary deep eutectic solvent according to claim 4, characterized in that In the ternary deep eutectic solvent, the molar ratio of component A, component B and component C is (8-12):(8-12):

1.

6. A method for preparing the ternary deep eutectic solvent according to claim 1, characterized in that: The preparation method comprises the following steps: Mixing component A and component B, and performing a first heating and stirring process to obtain a mixed solution; Component C is added to the mixed solution, and a second heating and stirring treatment is performed to obtain the ternary deep eutectic solvent.

7. The method for preparing a ternary deep eutectic solvent according to claim 6, wherein: The temperature of the first heating and stirring treatment is 50-80° C., and the time is 1-3 hours.

8. The method for preparing a ternary deep eutectic solvent according to claim 6, wherein: The temperature of the second heating and stirring treatment is 35-90° C., and the time is 3-6 hours.

9. Use of the ternary deep eutectic solvent according to any one of claims 1 to 5 or the ternary deep eutectic solvent prepared by the preparation method according to any one of claims 6 to 8 in the preparation of a drug.

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