A deep eutectic solvent and a preparation method and application thereof

Abstract

The application belongs to the technical field of low eutectic solvent and specifically relates to a low eutectic solvent and a preparation method and application thereof, the low eutectic solvent comprising hydroxytyrosol and cnidium, the molar ratio of the hydroxytyrosol and the cnidium being (1-9):(9-1), and the preparation method of the low eutectic solvent comprising the following steps: (1) washing a reactor, drying treatment, opening stirring, heating, the heating temperature being 50-70 DEG C, and inert gas protection; (2) adding the hydroxytyrosol and the cnidium in proportion, keeping stirring and inert gas protection after melting, and continuously keeping warm for 1-2 hours to prepare the low eutectic solvent, the low eutectic solvent being prepared into supermolecular microcapsules to obtain supermolecular microcapsule bacteriostatic ointment, the bacteriostatic ointment being applied to skin bacteriostatic drugs or daily chemicals, promoting skin penetration of hydroxytyrosol and reducing irritation.

Description

Technical Field

[0001] This invention belongs to the field of eutectic solvent technology, specifically relating to a eutectic solvent, its preparation method, and its application. Background Technology

[0002] Deep eutectic solvents (DES) are organic solvents with thermodynamic properties similar to those of ILs. DES consists of two main components: hydrogen bond donors (HBDs), such as acids, amines, or alcohols, and hydrogen bond acceptors (HBAs), such as tetraalkylammonium, quaternary ammonium salts, or phosphonium salts.

[0003] Both skin infections and skin lesions can lead to skin diseases, with skin infections causing more common skin diseases. Skin infections are mainly caused by bacteria or fungi, such as Staphylococcus aureus, Escherichia coli, and Candida albicans. Therefore, the clinical treatment of skin diseases caused by skin infections generally employs disinfection and antibacterial methods. Existing disinfectant skin medications mainly use antibacterial ingredients, such as chlorhexidine and miconazole. However, these antibacterial drugs have irritating effects and are not suitable for sensitive skin. Summary of the Invention

[0004] To address the problems in the prior art, this invention provides a low-eutectic solvent, its preparation method, and its application. This achieves the goal of using natural antibacterial raw materials hydroxytyrosol and osthol to prepare a supramolecular antibacterial DES (hydroxytyrosol-osthol) with synergistic and penetration-enhancing effects. Furthermore, by utilizing this DES in conjunction with supramolecular microencapsulation technology, an antibacterial ointment is prepared, further improving the antibacterial and penetration-enhancing efficiency of Cnidium monnieri while significantly reducing irritation.

[0005] The technical problem solved by this invention is achieved by the following technical solution:

[0006] A eutectic solvent comprising hydroxytyrosol and osthol, wherein the molar ratio of hydroxytyrosol to osthol is (1-9):(9-1).

[0007] Osthol, also known as methoxycarpus methyl ether, is a natural coumarin derivative extracted from Cnidium monnieri. It has a wide range of pharmacological functions, such as broad-spectrum antibacterial, anti-inflammatory, antioxidant, and anticancer effects. In terms of agricultural bioactivity, osthol, as an insecticide and fungicide, can inhibit the growth of various plant pathogens and has a good control effect on various crop pests. Its mechanism of action is to affect the growth of fungal cell walls, leading to a large number of hyphae breakage, while inhibiting the growth of pathogen hyphae. Furthermore, it has low toxicity and is easily decomposed in nature.

[0008] Hydroxytyrosol is a phenolic compound extracted from olive trees and their leaves. It has antioxidant, anti-atherosclerotic, antithrombotic, antibacterial, anti-inflammatory and antitumor effects.

[0009] Furthermore, the molar ratio of hydroxytyrosol to osthol is (1-4):(4-1).

[0010] Furthermore, a method for preparing a eutectic solvent includes the following steps:

[0011] (1) Clean the reactor, dry it, turn on the stirring and heating, and heat it at 50-70℃. Inert gas is introduced for protection.

[0012] (2) Add hydroxytyrosol and osthol in proportion, and after melting, keep stirring and inert gas protection, and continue to keep warm for 1-2 hours to obtain eutectic solvent.

[0013] DES is prepared by heating a mixture of HBA and HBD until a homogeneous, transparent solution is formed, or by dissolving the individual components in a solvent and then evaporating the solvent. The melting point of DES is significantly lower than that of its individual components.

[0014] The application of a eutectic solvent in the preparation of antibacterial ointment, wherein the antibacterial ointment is prepared by supramolecular microencapsulation technology to obtain supramolecular microencapsulated antibacterial ointment.

[0015] Supramolecular microencapsulation technology is built upon liposomes, microcapsules, ionic liquid stabilization technology, dermal cell-targeted release technology, hair follicle-targeted release technology, and inflammatory factor-responsive release technology. The product precisely delivers effective active ingredients by creating artificial transport channels.

[0016] Furthermore, the supramolecular microcapsule antibacterial ointment comprises the following components by weight percentage: hydroxytyrosol-osthol eutectic solvent: 0.1-5%; emulsifier: 1-5%; stabilizer: 0.2-2%; phospholipid: 0.5-5%; humectant: 5-20%; purified water: balance.

[0017] The emulsifier is selected from at least one of the following: PEG-40 hydrogenated castor oil, Tween 80, PEG-100 glyceryl stearate, glyceryl stearate, stearyl alcohol polyether-21, sorbitol polyether-30 tetraoleate, cetearyl glucoside, cetearyl alcohol, polyglycerol-10 laurate, behenol.

[0018] The stabilizer component is selected from at least one of the following: EDTA-2Na, carbomer, xanthan gum, sodium polyacrylate, hydroxypropyl cyclodextrin, acrylate / C10-30 alkanol acrylate crosspolymer.

[0019] The phospholipid component is selected from at least one of the following: lecithin, soybean lecithin, hydrogenated lecithin, and egg yolk lecithin.

[0020] The moisturizing ingredients are selected from at least one of the following: glycerin, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, ethoxydiethylene glycol, xylitol, betaine, pentylene glycol, and hexanediol.

[0021] Furthermore, the preparation method of the supramolecular microcapsule antibacterial ointment includes the following steps:

[0022] (1) Preparation of oil phase: Weigh hydroxytyrosol-osthol eutectic solvent, emulsifier and phospholipid, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain oil phase;

[0023] (2) Preparation of aqueous phase: Weigh out the humectant, stabilizer and water, heat to 60-80℃, stir to dissolve and mix thoroughly to obtain the aqueous phase;

[0024] (3) Shearing preparation: The oil phase obtained above is added to the aqueous phase and sheared at 5000-12000 rpm for 2-4 min to obtain supramolecular microcapsule antibacterial ointment pre-emulsion;

[0025] (4) Homogenization: The colostrum obtained above is homogenized at 400-800 bar, 5-8 times, and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0026] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0027] 1. This invention utilizes osthol, a naturally derived antibacterial raw material, to prepare natural DES, which exhibits stronger antibacterial activity and less irritation. In the pharmaceutical and daily chemical fields, further encapsulation using supramolecular microencapsulation technology yields an environmentally friendly, skin-friendly, and highly effective supramolecular microencapsulated antibacterial ointment.

[0028] 2. When the molar ratio of hydroxytyrosol to osthol DES prepared in this invention is between 4:1 and 1:4, it has a synergistic effect on antibacterial efficiency; secondly, the supramolecular microcapsule antibacterial ointment prepared by combining supramolecular microcapsule technology can further improve the antibacterial efficiency; the supramolecular microcapsule antibacterial ointment has lower irritation.

[0029] 3. The preparation method of the present invention is simple and feasible to operate, easy to control, has high production efficiency, and can be mass-produced.

[0030] 4. In this invention, the hydroxytyrosol-osthol eutectic solvent has a more efficient antibacterial rate, which can reduce the dosage, reduce the amount used and increase the effect, and save material costs.

[0031] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above contents, objectives, features and advantages of the present invention more obvious and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0032] Figure 1 This is a diagram of the natural eutectic solvent for the hydroxytyrosol-osthol combination of the present invention.

[0033] Figure 2 This is a diagram of the hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment of the present invention.

[0034] Figure 3 DSC spectra of hydroxytyrosol, osthol, and hydroxytyrosol-osthol DES.

[0035] Figure 4 Hydroxytyrosol, osthol, and hydroxytyrosol-osthol DES 1 H-NMR spectrum.

[0036] Figure 5 Hydroxytyrosol-osthol supramolecular microcapsules Transmission electron micrograph of the antibacterial ointment.

[0037] Figure 6 Hydroxytyrosol-osthol supramolecular microcapsules Appearance stability test chart of antibacterial ointment.

[0038] Figure 7 This is a particle size stability test result for hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment.

[0039] Figure 8 The graph shows the experimental results of the supramolecular microcapsule antibacterial ointment of the present invention and the inhibitory rates of each comparative example against Staphylococcus aureus.

[0040] Figure 9 The graph shows the experimental results of the supramolecular microcapsule antibacterial ointment of the present invention and the inhibitory rates of various comparative examples against Candida albicans.

[0041] Figure 10 This is a graph showing the results of the hydroxytyrosol skin retention test. Detailed Implementation

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0043] In addition, unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or prepared by existing methods.

[0044] Example 1: Preparation of Hydroxytyrosol-Osthol DES

[0045] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 70°C, and introduce inert gas for protection;

[0046] (2) Add hydroxytyrosol and osthol (1 mol: 9 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 2 hours to obtain DES.

[0047] Example 2: Preparation of Hydroxytyrosol-Osthol DES

[0048] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 60°C, and introduce inert gas for protection;

[0049] (2) Add hydroxytyrosol and osthol (1 mol: 4 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 1.5 hours to obtain DES.

[0050] Example 3: Preparation of Hydroxytyrosol-Osthol DES

[0051] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 50°C, and introduce inert gas for protection;

[0052] (2) Add hydroxytyrosol and osthol (2 mol: 3 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 1 hour to obtain DES.

[0053] Example 4: Preparation of Hydroxytyrosol-Osthol DES

[0054] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 70°C, and introduce inert gas for protection;

[0055] (2) Add hydroxytyrosol: osthol (1 mol: 1 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 2 hours to obtain DES.

[0056] Example 5: Preparation of Hydroxytyrosol-Osthol DES

[0057] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 60°C, and introduce inert gas for protection;

[0058] (2) Add hydroxytyrosol and osthol (3 mol: 2 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 1 hour to obtain DES.

[0059] Example 6: Preparation of Hydroxytyrosol-Osthol DES

[0060] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 60°C, and introduce inert gas for protection;

[0061] (2) Add hydroxytyrosol and osthol (4 mol: 1 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 2 hours to obtain DES.

[0062] Example 7: Preparation of Hydroxytyrosol-Osthol DES

[0063] (1) Clean the reactor, dry it, turn on the stirring and heating, heat the temperature to 70°C, and introduce inert gas for protection;

[0064] (2) Add hydroxytyrosol and osthol (9 mol: 1 mol), and after melting, keep stirring and inert gas protection, and continue to keep warm for 1.5 hours to obtain DES.

[0065] Example 8: Preparation of a hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0066] (1) Preparation of the oil phase:

[0067] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.1%; PEG-40 hydrogenated castor oil 1%; soybean lecithin 0.5%;

[0068] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0069] (2) Preparation of aqueous phase:

[0070] Weigh out the following: glycerin: 3%, dipropylene glycol: 2%, carbomer: 0.2%, and water: 93.2%.

[0071] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0072] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 2 minutes at a speed of 5000 rpm to obtain the supramolecular microcapsule antibacterial ointment primary emulsion.

[0073] (4) High-pressure homogenization preparation of supramolecular microcapsules

[0074] Antibacterial ointment: The colostrum obtained above was homogenized under high pressure at 400 bar, cyclicated 5 times, and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0075] Example 9: Preparation of a hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0076] (1) Preparation of the oil phase:

[0077] Hydroxytyrosol-osthol (4mol:1mol) DES: 5%; Stearyl alcohol polyether-21: 2%, Tween 80: 3%; Phospholipids: Hydrogenated lecithin: 5%;

[0078] Heat to the set temperature of 80°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0079] (2) Preparation of aqueous phase:

[0080] Weigh out: glycerol: 5%, 1,3-propanediol: 15%; acrylate / C10-30 alkanol acrylate crosspolymer: 1.0%, xanthan gum: 0.95%, EDTA-2na: 0.05%; water: 63%;

[0081] Heat to the set temperature of 80°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0082] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above was added to the aqueous phase and sheared at high speed for 4 min at a speed of 12000 rpm to obtain the primary emulsion of supramolecular microcapsule antibacterial ointment.

[0083] (4) High pressure homogenization to prepare supramolecular microcapsule antibacterial ointment: The colostrum obtained above was homogenized at 800 bar for 8 cycles and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0084] Example 10: Preparation of a hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0085] (1) Preparation of the oil phase:

[0086] Hydroxytyrosol-osthol (4mol:1mol) DES: 2%; Sorbitol ether-30 tetraoleate: 2%; Cetearyl glucoside: 1%; Lecithin: 3%;

[0087] Heat to the set temperature of 70°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0088] (2) Preparation of aqueous phase:

[0089] Weigh out: glycerin: 5%, propylene glycol: 5%; acrylate / C10-30 alkyl acrylate crosspolymer: 0.5%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 81.25%;

[0090] Heat to the set temperature of 70°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0091] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm to obtain the supramolecular microcapsule antibacterial ointment primary emulsion.

[0092] (4) High pressure homogenization to prepare supramolecular microcapsule antibacterial ointment: The colostrum obtained above was homogenized at 600 bar for 6 cycles and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0093] Example 11: Preparation of a hydroxytyrosol-osthol supramolecular microcystis ointment

[0094] (1) Preparation of the oil phase:

[0095] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.2%; PEG-40 hydrogenated castor oil: 2%; cetearyl glucoside: 1%; lecithin: 2%;

[0096] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0097] (2) Preparation of aqueous phase:

[0098] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylic acid (acetate) / C10-30 alkyl acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 78.25%;

[0099] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0100] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm to obtain the supramolecular microcapsule antibacterial ointment primary emulsion.

[0101] (4) High pressure homogenization to prepare supramolecular microcapsule antibacterial ointment: The colostrum obtained above was homogenized at 600 bar for 6 cycles and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0102] Example 12: Preparation of a hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0103] (1) Preparation of the oil phase:

[0104] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.4%; PEG-40 hydrogenated castor oil: 2%; cetearyl glucoside: 1%; lecithin: 2%;

[0105] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0106] (2) Preparation of aqueous phase:

[0107] Glycerin: 8%, Propylene glycol: 8%; Acrylic acid esters / C10-30 alkanol acrylate crosspolymer: 0.3%, Xanthan gum: 0.2%, EDTA-2na: 0.05%; Water: 78.05%;

[0108] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0109] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm to obtain the supramolecular microcapsule antibacterial ointment primary emulsion.

[0110] (4) High pressure homogenization to prepare supramolecular microcapsule antibacterial ointment: The colostrum obtained above was homogenized at 600 bar for 6 cycles and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0111] Example 13: Preparation of a hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0112] (1) Preparation of the oil phase:

[0113] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.8%; PEG-40 hydrogenated castor oil: 2%; cetearyl glucoside: 1%; lecithin: 2%;

[0114] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0115] (2) Preparation of aqueous phase:

[0116] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylate / C10-30 alkyl acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 77.65%.

[0117] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0118] (3) High-speed shearing preparation of the primary emulsion: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm to obtain the supramolecular microcapsule antibacterial ointment primary emulsion.

[0119] (4) High pressure homogenization to prepare supramolecular microcapsule antibacterial ointment: The colostrum obtained above was homogenized at 600 bar for 6 cycles and cooled to room temperature to obtain supramolecular microcapsule antibacterial ointment.

[0120] Comparative Example 1: Preparation of a supramolecular ointment containing 0.2% hydroxytyrosol-osthol (4 mol: 1 mol) DES

[0121] (1) Preparation of the oil phase:

[0122] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.2%; PEG-40 hydrogenated castor oil: 2%; cetearyl glucoside: 1%.

[0123] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0124] (2) Preparation of aqueous phase:

[0125] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylate / C10-30 alkanol acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 0.25%.

[0126] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0127] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm, and then cooled to room temperature to obtain supramolecular ointment.

[0128] Comparative Example 2: Preparation of a supramolecular ointment containing 0.4% hydroxytyrosol-osthol (4 mol: 1 mol) DES

[0129] (1) Preparation of the oil phase:

[0130] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.4%; PEG-40 hydrogenated castor oil: 2%; Cetearyl glucoside: 1%;

[0131] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0132] (2) Preparation of aqueous phase:

[0133] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylic acid (acetate) / C10-30 alkanol acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 0.05%.

[0134] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0135] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm, and then cooled to room temperature to obtain supramolecular ointment.

[0136] Comparative Example 3: Preparation of a supramolecular ointment containing 0.8% hydroxytyrosol-osthol (4 mol: 1 mol) DES

[0137] (1) Preparation of the oil phase:

[0138] Hydroxytyrosol-osthol (4mol:1mol) DES: 0.8%; PEG-40 hydrogenated castor oil: 2%; Cetearyl glucoside: 1%;

[0139] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0140] (2) Preparation of aqueous phase:

[0141] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylic acid (acetate) / C10-30 alkanol acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 79.65%.

[0142] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0143] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm, and then cooled to room temperature to obtain supramolecular ointment.

[0144] Comparative Example 4: Preparation of a common ointment containing 0.2% hydroxytyrosol and osthol (in a ratio of 4 mol: 1 mol).

[0145] (1) Preparation of the oil phase:

[0146] Hydroxytyrosol: 0.1467%; Osthol: 0.0533%; PEG-40 hydrogenated castor oil: 2%; Cetearyl glucoside: 1%;

[0147] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0148] (2) Preparation of aqueous phase:

[0149] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylic acid (acetate) / C10-30 alkanol acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 0.25%.

[0150] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0151] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm. After cooling to room temperature, a common ointment is obtained.

[0152] Comparative Example 5: Preparation of a common ointment containing 0.4% hydroxytyrosol and osthol (in a ratio of 4 mol: 1 mol).

[0153] (1) Preparation of the oil phase:

[0154] Hydroxytyrosol: 0.2934%; Osthol: 0.1066%; PEG-40 hydrogenated castor oil: 2%; Cetearyl glucoside: 1%;

[0155] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0156] (2) Preparation of aqueous phase:

[0157] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylic acid (acetate) / C10-30 alkanol acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 0.05%.

[0158] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0159] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm. After cooling to room temperature, a common ointment is obtained.

[0160] Comparative Example 6: Preparation of a common ointment containing 0.8% hydroxytyrosol and osthol (in a ratio of 4 mol: 1 mol).

[0161] (1) Preparation of the oil phase:

[0162] Hydroxytyrosol: 0.5868%; Osthol: 0.2132%; PEG-40 hydrogenated castor oil: 2%; Cetearyl glucoside: 1%;

[0163] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the oil phase.

[0164] (2) Preparation of aqueous phase:

[0165] Weigh out: glycerol: 8%, propylene glycol: 8%; acrylate / C10-30 alkyl acrylate crosspolymer: 0.3%, xanthan gum: 0.2%, EDTA-2na: 0.05%; water: 79.65%.

[0166] Heat to the set temperature of 60°C, stir to dissolve and mix thoroughly to obtain the aqueous phase.

[0167] (3) Preparation of primary emulsion by high-speed shearing: The oil phase obtained above is added to the aqueous phase and sheared at high speed for 3 minutes at a speed of 9000 rpm. After cooling to room temperature, a common ointment is obtained.

[0168] Test Methods Section

[0169] 1. Determination of melting point using DSC differential scanning calorimetry

[0170] The hydroxytyrosol-osteoin DES, hydroxytyrosol monomer, and osteoin monomer obtained in Example 3 were subjected to nitrogen protection at a temperature of -50°C to 100°C and a heating rate of 10°C / min to obtain their DSC spectra.

[0171] The results are as follows Figure 3 As shown, the results indicate that the prepared hydroxytyrosol-osthol DES solvent is homogeneous and has a significantly reduced melting point.

[0172] 2. Detection by proton nuclear magnetic resonance (NMR) spectroscopy

[0173] Nuclear magnetic resonance spectrometer: Bruker, Switzerland, AVANCE NEO 400;

[0174] Frequency: 400MHz;

[0175] Injection volume: 5 mg;

[0176] Solvent: Deuterated DMSO

[0177] Each of the following 5 mg of osthol monomer, hydroxytyrosol monomer, and hydroxytyrosol-osthol DES obtained in Example 6 was dissolved in 0.6 mL of deuterated DMSO reagent and detected by 1H NMR spectroscopy.

[0178] The result is as follows Figure 4 As shown, the results indicate that the hydrogen peak position of the eutectic solvent is shifted compared to that of each monomer, proving that the eutectic solvent is a new eutectic substance rather than a physical mixture.

[0179] 3. Observation using transmission electron microscopy

[0180] The hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment obtained in Example 12 was diluted with distilled water. The sample was then aspirated via capillary tube and blown onto a support grid. After staining, washing, and drying, the sample was observed using a transmission electron microscope, and the results were photographed and recorded. The results are as follows: Figure 5 As shown.

[0181] As shown in the figure, the nanoparticles of the prepared hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment are uniformly dispersed, and are round or elliptical in shape with a particle size of about 200 nm.

[0182] 4. Stability testing of hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment

[0183] Light stability chamber: STIK drug stability test chamber IMT-250L;

[0184] Thermal stability chamber: Boxun Pharmaceutical Stability Test Chamber BXY-2501;

[0185] 5℃ Refrigerated Box: Haier Laboratory Explosion-proof Refrigerated Box HLR-310FL;

[0186] -13℃ Freezer: Midea BD / BC-203KMD(E);

[0187] Nanoparticle size zeta potential analyzer: Anton Paar Litesizer 500.

[0188] Experimental Method: Example 12 was dispensed into 15ml transparent PE bottles and placed in a light chamber at 45℃, 5℃, -13℃, 25℃, and after 7 days, 14 days, 28 days, and 2 months. The appearance and particle size were then observed. Specific results are as follows: Figure 6 , 7 .

[0189] The results showed that the hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment had good stability.

[0190] 5. Antibacterial test

[0191] 5.1 Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

[0192] Test method:

[0193] (1) Add 3.0-5.0 mL of physiological saline to the cultured bacterial slant and scrape off the bacterial growth with a sterile cotton swab. Transfer the bacterial suspension to a sterile test tube using a pipette, dilute the bacterial suspension with physiological saline, shake to mix, and then measure the absorbance using a quartz cuvette. Select a bacterial suspension with a suitable absorbance value and store the prepared bacterial suspension at 4℃ for later use.

[0194] (2) The sample was prepared as a stock solution of 10 mg / ml in Luria-Bertani (LB) liquid medium. It was then diluted to the appropriate concentration using the half-dilution method in test tubes.

[0195] (3) Add 50 μL of bacterial suspension with a concentration of 10⁶ CFU / mL to each tube. Take two additional tubes as a negative control (without bacteria) and a blank control (physiological saline). Place them on a 37℃ constant temperature shaker and shake (180 rpm) for 24 h. Compare the negative control and blank control tubes and observe the degree of turbidity. The concentration corresponding to the first tube that does not show turbidity is the minimum inhibitory concentration (MIC).

[0196] (4) Take 100 μL of liquid from all the non-turbid test tubes and spread it evenly on LB solid medium. Continue to incubate in a 37°C incubator for 24 hours. The concentration corresponding to the plate with less than 5 colonies is the minimum bactericidal concentration (MBC).

[0197] (5) Repeat the above experiment 3 times and calculate the average value.

[0198] The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of hydroxytyrosol monomer, osthol monomer, and hydroxytyrosol-osthol DES against Staphylococcus aureus and Candida albicans were determined using the above methods. The specific results are shown in Tables 1 and 2 below:

[0199] Table 1

[0200]

[0201] Table 2

[0202]

[0203] The results showed that hydroxytyrosol-osthol DES exhibited synergistic inhibitory effects on Staphylococcus aureus and Candida albicans at ratios of 4 mol:1 mol, 3 mol:2 mol, 2 mol:3 mol, and 1 mol:4 mol. The optimal ratio of hydroxytyrosol-osthol DES for inhibiting Staphylococcus aureus and Candida albicans was 4 mol:1 mol.

[0204] 5.2 Antibacterial rate test

[0205] Test method:

[0206] (1) Add 3.0-5.0 mL of physiological saline to the cultured bacterial slant, and scrape off the bacterial growth with a sterile cotton swab. Transfer the bacterial suspension to a sterile test tube using a pipette, dilute the bacterial suspension with physiological saline, shake to mix, and then measure the absorbance using a quartz cuvette. Select a bacterial suspension with a suitable absorbance value and store the prepared bacterial suspension in a 4 ℃ refrigerator for later use.

[0207] (2) Take 0.1 mL of the diluted bacterial solution and add it to a 5 mL sample tube. Mix quickly and start timing for 5 min immediately.

[0208] (3) After the specified time has elapsed, take 0.5 mL of the mixture of test bacteria and sample and add it to a test tube containing 4.5 mL of sterile PBS, and mix thoroughly;

[0209] (4) After standing for 10 min, take 0.1 mL of the sample solution (or after appropriate dilution, take 2-3 dilutions) into a blood agar plate and spread it using a sterile spreader, or take 1 mL of the sample solution into a sterile Petri dish and pour 15 mL of MRS agar medium cooled to 40-45℃ into the dish, rotate the dish to make it fully uniform, and make 2 replicates for each dilution. After the agar solidifies, turn the Petri dish over and incubate in an anaerobic incubator at 37℃ for 48 h, and then count the viable colonies.

[0210] (5) Replace the test sample with PBS, take 0.2 mL and follow the above steps as a control sample;

[0211] (6) The experiment was repeated 3 times, the average value was calculated, and the antibacterial rate was calculated.

[0212] Calculation formula:

[0213] Antibacterial rate (%) = ×100

[0214] The inhibition rates of hydroxytyrosol-osthol (4 mol: 1 mol) supramolecular microcapsule antibacterial ointments (Examples 11, 12, 13), supramolecular antibacterial ointments (Comparative Examples 1, 2, 3), and ordinary ointments at concentrations of 0.5 MIC, 1 MIC, and 2 MIC against Staphylococcus aureus and Candida albicans were determined using the above methods. The results are shown in Tables 3 and 4 below:

[0215] Table 3

[0216]

[0217] Table 4

[0218]

[0219] From the table above, we can obtain... Figure 8 , 9 The results showed that the antibacterial ability of hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment was significantly enhanced compared with that of supramolecular ointment against Staphylococcus aureus and Candida albicans; and the antibacterial ability of supramolecular ointment was significantly enhanced compared with that of ordinary antibacterial ointment against Staphylococcus aureus and Candida albicans.

[0220] Therefore, it can be seen that:

[0221] (1) Supramolecular DES can effectively enhance the antibacterial ability of hydroxytyrosol and osthol, and has a synergistic effect.

[0222] (2) The supramolecular microencapsulation technology further enhances the antibacterial ability of hydroxytyrosol and osthol DES.

[0223] 6. Repeated skin irritation tests

[0224] 6.1 Experimental Methods

[0225] (1) Animals were allowed to adapt to the experimental animal room environment for 3 days before the experiment to ensure that the animals entering the group were healthy and had no broken skin.

[0226] (2) 24 hours before the experiment, the hair on both sides of the spine on the back of the experimental animal was shaved off. The hair removal area was about 3cm x 3cm on the left and right sides, and the skin was not damaged. The area covered by the smear was 2.5cm x 2.5cm.

[0227] (3) After weighing the sample the next day, apply 0.5 mL of the test substance to the left side of the skin. The right side serves as a blank control. Apply once a day for 14 consecutive days.

[0228] (4) Clinical observation: Starting from the second day, remove the residual test substance with warm water and observe the results after 1 hour.

[0229] (5) The judgment criteria and scoring shall be based on the results of the skin irritation / corrosiveness test 5.4.3 in Chapter 6 of the Chemical Safety Technical Specification (2015 edition). Multiple skin irritation reaction scores shall be conducted, and the erythema and edema of the samples shall be observed and scored. The average score of each animal per day shall be calculated to judge the intensity of skin irritation.

[0230] Using the methods described above, we conducted multiple skin irritation tests on Example 12, Comparative Example 2, and Comparative Example 5. The specific results are shown in Table 5 below.

[0231] Table 5

[0232]

[0233] The results showed that the hydroxytyrosol-osthol supramolecular microcapsule antibacterial ointment could significantly reduce the skin irritation of hydroxytyrosol-osthol.

[0234] 7. Method for detecting hydroxytyrosol content

[0235] Chromatographic conditions:

[0236] The chromatographic column was an Agilent TC-C18 (4.6 mm × 250 mm, 5 μm); the mobile phase was 0.12% formic acid aqueous solution: 0.12% formic acid methanol solution = 85:15 (V:V); the detection wavelength was 280 nm; the column temperature was 30 °C; and the injection volume was 20 μl.

[0237] Hydroxytyrosol standard curve:

[0238] Weigh an appropriate amount of hydroxytyrosol into a brown volumetric flask, dissolve it in methanol, dilute to volume, and prepare a concentration gradient solution. Analyze the solution using HPLC. Plot a standard curve with the mass concentration of hydroxytyrosol on the x-axis and the peak area on the y-axis, and perform linear regression. The regression equation is y = 187.62x + 48.61 (R² = 0.9996), showing good linearity in the range of 0.5–100 μg / mL.

[0239] 8. Skin Retention Volume Study

[0240] Before the experiment, a pigskin model with a thickness of 300±50 μm was prepared using a skin grafting scalpel. This model was then cut into small circular pieces the size of the receiving pool and placed in physiological saline for later use. Phosphate buffer (pH = 7.4) was used as the receiving medium. During the experiment, the skin model was fixed between the release pool and the receiving pool, with the stratum corneum side facing the release pool and the dermis side facing the receiving pool, ensuring close contact between the skin and the receiving solution, and preventing air bubbles from forming. Subsequently, a certain amount of the hydroxytyrosol-osteoin supramolecular microcapsule antibacterial ointment obtained in Example 12 and the hydroxytyrosol-osteoin supramolecular ointment obtained in Comparative Example 2 were applied to the skin surface, respectively. The temperature of the receiving pool was maintained at 37±0.5℃, and a magnetic ball was placed inside the receiving pool, rotating at 300 rpm throughout the experiment. The control group and the experimental group used the hydroxytyrosol-osteoin ordinary ointment obtained in Comparative Example 5. Three parallel samples were set up for both the control and experimental groups.

[0241] Eight hours after the in vitro percutaneous treatment, the skin was removed, and the residual solution on the surface was rinsed off with pure water. The skin from the transdermal site was taken, minced, and homogenized with 1.5 ml of ethanol. The mixture was sonicated for 1 hour to extract hydroxytyrosol from the skin. The homogenate was transferred to a centrifuge tube, vortexed, and centrifuged at 12,000 rpm for 10 minutes. The residue was extracted again with 1 ml of ethanol. The two supernatants were combined, mixed, and the supernatant was filtered through a 0.22 μm filter membrane and analyzed by liquid chromatography to determine the amount of hydroxytyrosol retained in the skin.

[0242] Test results are as follows Figure 10As shown in the results, the amount of hydroxytyrosol retained in the skin in Example 12 was 4.579 times that in Comparative Example 5, and the amount of hydroxytyrosol retained in the skin in Comparative Example 2 was 2.106 times that in Comparative Example 5. This indicates that the hydroxytyrosol osthol DES in this invention has a penetration-enhancing effect, and the hydroxytyrosol osthol supramolecular microcapsules can further enhance the penetration-enhancing effect.

[0243] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0244] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A deep eutectic solvent characterized in that, Prepared from hydroxytyrosol and cnidilide, the molar ratio of hydroxytyrosol and cnidilide is (1-9):(9-1); The preparation method comprises the following steps: (1) Wash the reactor, dry it, turn on the stirring and heat it to a temperature of 50-70℃, and then protect it with inert gas; (2) Add hydroxytyrosol and cnidilide in proportion, keep stirring and inert gas protection after melting, and continue to keep warm for 1-2 hours to obtain the eutectic solvent.

2. The deep eutectic solvent of claim 1, wherein: The molar ratio of hydroxytyrosol and cnidilide is (1-4):(4-1).

3. Use of a deep eutectic solvent according to claim 1 or 2 for the preparation of an antiseptic ointment, characterized in that: The super-molecular micro-capsule bacteriostatic ointment is prepared by the super-molecular micro-capsule technology.

4. Use of the deep eutectic solvent according to claim 3 for the preparation of an antiseptic ointment, characterized in that: The super-molecular micro-capsule bacteriostatic ointment comprises the following components in mass percentage: hydroxytyrosol-cnidilide eutectic solvent: 0.1-5%; emulsifier: 1-5%; stabilizer: 0.2-2%; phospholipid: 0.5-5%; humectant: 5-20%; and pure water: the balance. The emulsifier is at least one of PEG-40 hydrogenated castor oil, Tween 80, PEG-100 glyceryl stearate, glyceryl stearate, steareth-21, sorbeth-30 tetraoleate, cetearyl glucoside, cetearyl alcohol, polyglyceryl-10 laurate, and behenyl alcohol; the stabilizer is at least one of EDTA-2Na, carbomer, xanthan gum, sodium polyacrylate, hydroxypropyl cyclodextrin, and acrylic acid (ester) / C10-30 alkanol acrylate cross-linked polymer; the phospholipid is at least one of lecithin, soybean lecithin, hydrogenated lecithin, and egg yolk lecithin; and the humectant is at least one of glycerol, butylene glycol, propylene glycol, 1,3-propanediol, dipropylene glycol, ethoxydiglycol, xylitol, betaine, pentylene glycol, and hexylene glycol. The preparation method of the super-molecular micro-capsule bacteriostatic ointment comprises the following steps:

5. Use of a deep eutectic solvent according to claim 4 for the preparation of an antiseptic ointment, characterized in that: (1) Prepare the oil phase: weigh the hydroxytyrosol-cnidilide eutectic solvent, emulsifier, and phospholipid, heat them to 60-80℃, stir and dissolve them sufficiently, and then mix them uniformly to obtain the oil phase; 6. Use of a deep eutectic solvent according to claim 5 for the preparation of an antiseptic ointment, characterized in that: (2) Prepare the water phase: weigh the humectant, stabilizer, and water, heat them to 60-80℃, stir and dissolve them sufficiently, and then mix them uniformly to obtain the water phase; (3) Shearing preparation: add the oil phase obtained above into the water phase, shear at a speed of 5000-12000 rpm for 2-4 min to obtain the super-molecular micro-capsule bacteriostatic ointment initial emulsion; (4) Homogenization: homogenize the initial emulsion obtained in (3) at 400-800 bar, circulate for 5-8 times, and then cool it to room temperature to obtain the super-molecular micro-capsule bacteriostatic ointment. ​ ​

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