Water-in-water eutectic solvent microemulsion and preparation method thereof

Abstract

The present invention relates to the field of pharmaceutics, and discloses a water-in-water deep eutectic solvent microemulsion, which comprises: a hydrophobic deep eutectic solvent, which is prepared by mixing at least two components according to a specific ratio, and the two components are respectively a terpenoid solvent and a fatty acid solvent; an emulsifier; an aqueous phase comprising an emulsifier and water; wherein the microemulsion is an oil-in-water type microemulsion, and the average particle size of the microemulsion is less than 300nm; the hydrophobic deep-eutectic solvent is a solvent prepared by mixing a diterpenoid solvent and a fatty acid solvent according to a weight ratio of 1: 9 to 9: 1. By adopting the deep eutectic solvent technology, the hydrophobic deep eutectic solvent is used for replacing a traditional oil solvent, the solubility of the quercetin is effectively improved, and the highest drug loading capacity of the quercetin in the preferable hydrophobic deep eutectic solvent can reach 23 mg / mL and is improved by 2000-3000 times compared with the highest drug loading capacity of the quercetin in water which is only 1 microgram / mL.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutics, and specifically relates to a water-in-deep eutectic solvent microemulsion and a preparation method thereof. Background Art

[0002] Quercetin (Que) is a flavonoid compound widely present in plants and has various pharmacological effects. Pharmacological studies have shown that Que has pharmacological effects such as antioxidant, anti-inflammatory, antibacterial, anti-cancer, and blood glucose regulation. However, its solubility in water is only about 1 μg / mL, and Quercetin has a strong first-pass effect, poor oral absorption effect, and low bioavailability (less than 17% in rats and less than 2% in humans), which greatly limits its pharmacological effects. Therefore, a large number of scholars have carried out research on Quercetin nano-formulations including nanoparticles, nanoemulsions, nanoliposomes, nanocrystals, nanocapsules, gold nanocages, nanotubes, etc.

[0003] The deep eutectic solvent system (DESs) provides a new idea for improving the solubility and permeability of active pharmaceutical ingredients. Deep eutectic solvents are produced by the physical mixing of two (solid) components in a certain proportion, which results in a decrease in the melting point. It is a new type of eutectic system. Different from traditional eutectic systems, it can reduce the melting point of the system to a greater extent, and its melting point is lower than room temperature and the melting points of the two components. Their thermodynamic stability makes them superior to amorphous drug delivery systems. As a promising drug delivery strategy, researchers have used DESs for various purposes, such as: (1) as a medium for the synthesis and processing of biopolymers and inorganic nanomaterials; (2) solubilization - improving the solubility of poorly soluble drugs; (3) enhancing permeability - applied in the fields of dermal, transdermal, oral, buccal, and nasal drug delivery systems. Therefore, we use hydrophobic DES as the oil phase, and form a microemulsion with the aqueous phase and emulsifier to improve the solubility of poorly soluble Quercetin in water, reduce the toxicity of the delivery system, promote drug penetration and absorption, and improve bioavailability.

[0004] Drug-loaded microemulsions are composed of oil droplets encapsulating bioactive ingredients. In the presence of surfactants, these oil droplets are well dispersed in the aqueous medium. It has been found that the pharmacological effects of drugs or bioactive compounds loaded into monodisperse emulsion droplets are closely related to the size and uniformity of the droplets. In addition, emulsions with a narrow particle size distribution are not prone to coalescence formed by the collision of large droplets and small droplets, and can effectively alleviate the problems of gravitational separation and Ostwald ripening of the emulsion. Therefore, strictly controlling the size of emulsion droplets is the key to ensuring the stability of the emulsion. Currently, the main emulsification preparation methods of DES microemulsions are stirring method, high-pressure homogenization method, ultrasonic method, etc. Taking the stirring method as an example, the formation of microemulsions depends on the shear mixing of immiscible liquids, but the reproducibility of the resulting uniform droplet size distribution is usually very poor, and the droplet size distribution is uneven. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a water-in-low eutectic solvent microemulsion and a preparation method thereof, which solve the problems of low solubility and low bioavailability of quercetin in the prior art.

[0006] To achieve the above objectives, the present invention is realized through the following technical solutions: A water-in-low eutectic solvent microemulsion, comprising: A hydrophobic low eutectic solvent, which is prepared by mixing at least two components in a specific ratio. The two components are a terpene solvent and a fatty acid solvent respectively; An emulsifier; An aqueous phase, which contains an emulsifier and water; Among them, the microemulsion is a water-in-oil microemulsion, and its average particle size is less than 300 nm.

[0007] Preferably, the hydrophobic low eutectic solvent is a solvent prepared by mixing a diterpene solvent and a fatty acid solvent in a weight ratio of 1:9 to 9:1.

[0008] Preferably, the terpene solvent is selected from at least one of the following, including DL-menthol, thymol, L-menthol, nerol, citronellol, citral, citronellal, hydroxycitronellal, phellandral, perillaldehyde, menthone, carvone, dihydrocarvone.

[0009] Preferably, the fatty acid solvent is selected from at least one of the following, including octanoic acid, decanoic acid, oleic acid, linoleic acid, myristic acid, lauric acid, palmitic acid, arachidic acid, linolenic acid, isolenic acid.

[0010] Preferably, the emulsifier is selected from at least one of the following, including Tween20, Tween80, EL-35, sodium dodecyl sulfate, Span20, Span40, polyoxyethylene fatty alcohol ether, poloxamer.

[0011] Preferably, the concentration of the emulsifier in the aqueous phase is 0.5% to 5%.

[0012] Preferably, the particle size distribution of the microemulsion is 300 nm and below, and the PDI value is less than 0.3.

[0013] Preferably, the drug loading of the water-in-low eutectic solvent microemulsion is 0.1 mg / mL and above.

[0014] Furthermore, the quercetin microemulsion has the following characteristics: (1) Average particle size: 300 nm and below; (2) Drug content: 0.1 mg / mL and above.

[0015] Specifically, the quercetin is a flavonoid compound with the chemical name 3,3’,4’,5,7-pentahydroxyflavone.

[0016] Specifically, in the composition of the hydrophobic eutectic solvent, the diterpene solvents are selected from at least one of the following: DL-menthol, thymol, L-menthol, nerol, citronellol, citral, citronellal, hydroxycitronellal, phellandral, perillaldehyde, menthone, carvone, dihydrocarvone, etc.; the fatty acid solvents are selected from at least one of the following: octanoic acid, decanoic acid, oleic acid, linoleic acid, myristic acid, lauric acid, palmitic acid, arachidic acid, linolenic acid, isooic acid, etc. The two can form a stable hydrophobic eutectic solvent through hydrogen bonding under water bath heating, with good biocompatibility and safety, and quercetin has a high solubility in the combined hydrophobic eutectic solvent.

[0017] Further, the hydrophobic eutectic solvent is composed of diterpene and fatty acid solvents, and the weight ratio is diterpene: fatty acid = 1:9 - 9:1, preferably DL-menthol: decanoic acid = 7:3 - 9:1. Under this ratio, the formed hydrophobic eutectic solvent can dissolve the maximum amount of quercetin to increase the drug loading.

[0018] Preferably, the emulsifier is selected from any one of Tween80, Tween20, EL-35, sodium dodecyl sulfate, Span20, Span40, polyoxyethylene fatty alcohol ether, poloxamer, etc., and emulsifier Tween20 is preferred. Tween20, whose chemical name is polysorbate-20, is an oil-in-water (O / W) type emulsifier, with characteristics such as low toxicity, low irritation, and low conductivity. It can effectively reduce the surface tension and the repulsive force between micelles of the microemulsion, and improve the stability and solubilization ability of the microemulsion.

[0019] Further, the aqueous solution is composed of more than 0.5% emulsifier and water, preferably 1% - 5% emulsifier. The appropriate amount of emulsifier can improve the biocompatibility of the microemulsion and improve the stability of the microemulsion.

[0020] Further, in the blank microemulsion, based on volume, the volume ratio of each component is: 5% - 20% hydrophobic eutectic solvent, and the balance is the aqueous phase.

[0021] A preparation method of an oil-in-water eutectic solvent microemulsion includes the following steps: Prepare the hydrophobic eutectic solvent: Heat and mix the diterpene solvent and the fatty acid solvent in a specific ratio to obtain the hydrophobic eutectic solvent; Prepare the aqueous phase: Mix the emulsifier and water to obtain the aqueous phase; Mix the hydrophobic eutectic solvent and the aqueous phase through a microfluidic device to obtain the oil-in-water eutectic solvent microemulsion.

[0022] Preferably, the microfluidic device consists of a sampling syringe and a microchannel chip. The microchannel chip is made of PDMS or glass and has an inner diameter of 50 μm to 200 μm. The flow rate ratio of the hydrophobic deep eutectic solvent to the aqueous phase in the sampling syringe is 1:5 to 1:20.

[0023] Specifically, the prepared aqueous phase and hydrophobic deep eutectic solvent are added into the sampling syringe of the microfluidic device. By controlling the flow rate, the hydrophobic deep eutectic solvent is sheared by the aqueous phase to obtain a DES / W blank microemulsion.

[0024] The present invention provides a water-in-deep eutectic solvent microemulsion and a preparation method thereof, having the following beneficial effects: 1. By adopting the deep eutectic solvent technology, the present invention replaces the traditional oil solvent with a hydrophobic deep eutectic solvent, effectively improving the solubility of quercetin. The highest drug loading amount of quercetin in the preferred hydrophobic deep eutectic solvent can reach 23 mg / mL, compared with only 1 μg / mL in water, which is increased by 2000 - 3000 times.

[0025] 2. Through the microemulsion technology, the present invention can improve the penetration ability of quercetin in mucous membranes and tissues, making it more easily absorbed and utilized, thereby significantly improving the bioavailability of quercetin and enhancing the therapeutic effect of the drug.

[0026] 3. Based on the application of microfluidic technology, the present invention can effectively control the particle size distribution of the water-in-deep eutectic solvent microemulsion (DES / W microemulsion), thereby ensuring that the prepared microemulsion has high stability and repeatability, and further enhancing the reliability of its use as a drug delivery system. Description of the Drawings

[0027] Figure 1 It is the full wavelength scanning diagram of quercetin - ethanol solution; Figure 2 It is the linear relationship between quercetin concentration and ultraviolet absorption; Figure 3 It is the microemulsion particle size diagram when the emulsifier is Tween 20; Figure 4 It is the microemulsion particle size diagram when the emulsifier is Tween 80; Figure 5 It is the microemulsion particle size diagram when the emulsifier is EL - 35; Figure 6 It is the relationship diagram between the particle size and PDI of the microemulsion and the emulsifier concentration; Figure 7 It is the microemulsion particle size diagram of Example 1; Figure 8 It is the microemulsion particle size diagram of Example 2; Figure 9 Microemulsion particle size diagram for Example 3; Figure 10 Microemulsion particle size diagram for Example 4. Detailed implementation manners

[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0029] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following embodiments are all commercially available products unless otherwise specified.

[0030] Experimental steps: 1. Establishment of the analysis method for quercetin (1) Weigh quercetin precisely and prepare a curcumin solution with a certain concentration. Scan it in the wavelength range of 200 - 900 nm under an ultraviolet spectrophotometer to determine the maximum absorption wavelength of quercetin, as shown in Figure 1 .

[0031] (2) Preparation of the standard curve of the quercetin solution Weigh curcumin precisely and dissolve it in absolute ethanol to prepare solutions with concentrations of 1, 2, 3, 4, 5, and 6 μg / mL. Measure the absorbance of each concentration at the maximum ultraviolet absorption wavelength of 374 nm, and plot the absorbance-concentration standard curve, as shown in Figure 2 .

[0032] (3) Investigation of the solubility of quercetin Weigh 1 g of different liquids into different vials. Add an excessive amount of quercetin to them, vortex and mix well, then stir at a constant temperature of 100 r / min in the dark for 24 h. After reaching equilibrium, let it stand overnight. Take an appropriate amount of the liquid in the vial and put it into a 2 mL centrifuge tube, centrifuge at 4000 r / min for 15 min, take the supernatant and dilute it with absolute ethanol by a certain multiple, measure the absorbance at a wavelength of 374 nm using an ultraviolet spectrophotometer, and calculate the quercetin concentration.

[0033] 2. Selection of hydrophobic deep eutectic solvents According to the above method for investigating the solubility of quercetin, the results are shown in Table 1. As can be seen from the table, the solubility of quercetin shows a regular increasing trend in the above solvents. Therefore, hydrophobic deep eutectic solvents with a ratio of DL-menthol: n-decanoic acid = 7:3 - 9:1 are initially selected as the oil phase.

[0034] Table 1 Solubility of Quercetin in Different DESs Ratio DL-Menthol: Capric acid (mg / mL) Thymol: Capric acid (mg / mL) 1:9 / / 2:8 / / 3:7 0.902 0.058 4:6 1.054 0.061 5:5 1.447 0.087 6:4 1.572 0.084 7:3 2.437 0.074 8:2 3.143 0.084 9:1 3.158 0.086 3. Selection of Emulsifiers Please refer to the appendix Figure 3 - appendix Figure 5 , take emulsifiers Tween 80, Tween 20, EL-35 and purified water to form a continuous phase containing 1-5% emulsifier, and prepare microemulsions with the dispersed phase (DES) at different flow rates by a microchannel syringe respectively. Taking the particle size and PDI of the obtained microemulsions as the analysis criteria, Tween 20 was preferably selected, as shown in Table 2.

[0035] Table 2 Particle Size and PDI of Microemulsions under Different Emulsifiers Type Particle size (nm) PDI Tween 20 Tween 80 EL-35 140.842394122 0.1810.4420.117 4. Selection of Emulsifier Ratio Take emulsifier Tween 20 and purified water to form a continuous phase, making it contain 0.5%, 1%, 1.5%, 2%, 2.5%, 3% of the emulsifier, and prepare microemulsions with the dispersed phase (DES) at different flow rates by a microchannel syringe respectively. Taking the particle size and PDI of the obtained microemulsions as the analysis criteria, the preferred ratio of Tween 20 is 1% - 2.5%, see Figure 6 .

[0036] Example 1 A DES / W Microemulsion Please refer to the appendix Figure 7 , the preparation method is as follows: (1) Preparation of hydrophobic eutectic solvent: Weigh precisely DL-menthol and n-decanoic acid with a molar ratio of 7:3 - 9:1, heat and melt them at 80 °C for 4 h, stir and mix evenly, and cool to room temperature to form a hydrophobic eutectic solvent; (2) Preparation of aqueous phase: Place 1 g of Tween 20 and 99 g of purified water in a beaker, and mix them evenly by magnetic stirring for 10 minutes; (3) Preparation of DES / W blank microemulsion: Add the prepared aqueous phase and hydrophobic eutectic solvent into the injection syringe of the microfluidic device, control the total flow rate at 30 mL / h, and the two-phase flow rate ratio at 10:1, so that the hydrophobic eutectic solvent is sheared by the aqueous phase to obtain a DES / W blank microemulsion. Example 2 A DES / W Microemulsion Please refer to the appendix Figure 8 , the preparation method is as follows: (1) Preparation of hydrophobic eutectic solvent: Weigh precisely DL-menthol and n-decanoic acid with a molar ratio of 7:3 - 9:1, heat and melt them at 80 °C for 4 h, stir and mix evenly, and cool to room temperature to form a hydrophobic eutectic solvent; (2) Preparation of aqueous phase: Place 1 g of Tween 20 and 99 g of purified water in a beaker, and mix them evenly by magnetic stirring for 10 minutes; (3) Preparation of DES / W blank microemulsion: Add the prepared aqueous phase and hydrophobic deep eutectic solvent into the injection syringe of the microfluidic device, control the total flow rate at 40 mL / h and the two-phase flow rate ratio at 12.5:1, so that the hydrophobic deep eutectic solvent is sheared by the aqueous phase to obtain the DES / W blank microemulsion.

[0037] Please refer to the appendix Figure 9 , Example 3 A quercetin DES / W microemulsion The preparation method is as follows: (1) Preparation of hydrophobic deep eutectic solvent: Weigh precisely DL-menthol and n-decanoic acid with a molar ratio of 7:3 - 9:1, heat and dissolve them at 80 °C for 4 h, stir and mix evenly, and cool to room temperature to form the hydrophobic deep eutectic solvent; (2) Preparation of aqueous phase: Place 1 g of Tween 20 and 99 g of purified water in a beaker, and mix them evenly by magnetic stirring for 10 minutes; (3) Preparation of DES / W blank microemulsion: Add the prepared aqueous phase and hydrophobic deep eutectic solvent into the injection syringe of the microfluidic device, control the total flow rate at 30 mL / h and the two-phase flow rate ratio at 10:1, so that the hydrophobic deep eutectic solvent is sheared by the aqueous phase to obtain the DES / W blank microemulsion.

[0038] (4) Preparation of quercetin DES / W microemulsion: Add 0.4 mg of quercetin to 2 mL of the blank DES / W microemulsion, and dissolve it by ultrasonic treatment to obtain it.

[0039] Example 4 A quercetin DES / W microemulsion Please refer to the appendix Figure 10 , The preparation method is as follows: (1) Preparation of hydrophobic deep eutectic solvent: Weigh precisely DL-menthol and n-decanoic acid with a molar ratio of 7:3 - 9:1, heat and dissolve them at 80 °C for 4 h, stir and mix evenly, and cool to room temperature to form the hydrophobic deep eutectic solvent; (2) Preparation of aqueous phase: Place 1 g of Tween 20 and 99 g of purified water in a beaker, and mix them evenly by magnetic stirring for 10 minutes; (3) Preparation of DES / W blank microemulsion: Add the prepared aqueous phase and hydrophobic deep eutectic solvent into the injection syringe of the microfluidic device, control the total flow rate at 40 mL / h and the two-phase flow rate ratio at 12.5:1, so that the hydrophobic deep eutectic solvent is sheared by the aqueous phase to obtain the DES / W blank microemulsion.

[0040] (4) Preparation of quercetin DES / W microemulsion: Add 0.3 mg of quercetin to 2 mL of the blank DES / W microemulsion, and dissolve it by ultrasonic treatment to obtain it.

[0041] Investigation of the particle size of the microemulsion in the test example At 25 °C, the particle size and PDI distribution of the microemulsion samples prepared in Examples 1-4 were measured using a Malvern laser particle size analyzer. The results are shown in Table 3.

[0042] Table 3 Results of microemulsion particle size measurement Example Average particle size (nm) PDI Quercetin content (mg / mL) 13 118.0173.9 0.1350.105 / 0.2 24 146.7187.6 0.1750.111 / 0.15 Table 3 results show that the DES / W microemulsions prepared in Examples 1-4 all had particle sizes less than 200 nm. Moreover, compared with the corresponding blank microemulsions (the microemulsions prepared in Examples 1 and 2), the particle size of the quercetin DES / W microemulsion slightly increased, and there was no significant difference in the change of PDI.

[0043] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A deep eutectic solvent microemulsion in water, characterized in that: include: A hydrophobic deep eutectic solvent, which is prepared by mixing at least two components in a specific ratio, wherein the two components are a terpene solvent and a fatty acid solvent; Emulsifiers; an aqueous phase comprising an emulsifier and water; Wherein, the microemulsion is an oil-in-water microemulsion, and its average particle size is less than 300nm.

2. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The hydrophobic low eutectic solvent is a solvent prepared by mixing a diterpene solvent and a fatty acid solvent in a weight ratio of 1:9 to 9:

1.

3. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The terpene solvent is selected from at least one of the following: DL-menthol, thymol, L-menthol, nerol, citronellol, citral, citronellal, hydroxycitronellal, serraldehyde, perillaldehyde, menthone, carvone, and dihydrocarvone.

4. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The fatty acid solvent is selected from at least one of the following: caprylic acid, capric acid, oleic acid, linoleic acid, myristic acid, lauric acid, palmitic acid, arachidic acid, linolenic acid, and vaccenic acid.

5. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The emulsifier is selected from at least one of the following, including Tween 20, Tween 80, EL-35, sodium lauryl sulfate, Span 20, Span 40, polyoxyethylene fatty alcohol ether, and poloxamer.

6. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The concentration of the emulsifier in the aqueous phase is 0.5% to 5%.

7. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The particle size distribution of the microemulsion is 300 nm or less, and the PDI value is less than 0.

3.

8. The deep eutectic solvent microemulsion in water according to claim 1, characterized in that: The drug loading of the water-in-deep eutectic solvent microemulsion is 0.1 mg / mL or more.

9. A method for preparing a deep eutectic solvent microemulsion in water, according to any one of claims 1 to 8, characterized in that: The following steps are involved: Preparing a hydrophobic deep eutectic solvent: heating and mixing a diterpene solvent and a fatty acid solvent in a specific ratio to obtain a hydrophobic deep eutectic solvent; Preparing an aqueous phase: mixing an emulsifier with water to obtain an aqueous phase; The hydrophobic deep eutectic solvent is mixed with water through a microfluidic device to obtain a water-in-deep eutectic solvent microemulsion.

10. The method for preparing a deep eutectic solvent-in-water microemulsion according to claim 9, characterized in that: The microfluidic device is composed of a sample injection syringe and a microchannel chip, wherein the microchannel chip is made of PDMS or glass and has an inner diameter of 50 μm to 200 μm; The ratio of the flow rate of the hydrophobic deep eutectic solvent to the flow rate of the water phase in the injection syringe is 1:5 to 1:20.

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