Antibacterial composition containing olive leaf extract as well as preparation method and application of antibacterial composition

Abstract

The invention discloses an olive leaf extract-containing antibacterial composition and a preparation method and application thereof.The preparation method comprises the following steps: taking ferroferric oxide particles wrapped by oleic acid as a magnetic source, preparing magnetic polymer microspheres by a microemulsion method, coating the surfaces of the magnetic microspheres with silicon dioxide by a surface chemical coating modification method, and then performing amination modification to obtain the olive leaf extract-containing antibacterial composition. The obtained aminated magnetic microspheres are used for separating and purifying bacteriostatic active ingredients in a mixed solution with olive leaf extract as a main material and extracts of radix sophorae flavescentis, cortex phellodendri, fructus cnidii, liquorice, radix isatidis, honeysuckle, aloe and the like as auxiliary materials, and after desorption, a desorption solution is subjected to rotary evaporation concentration and vacuum drying; and compounding with several of nicotinamide, coenzyme Q10, nootkatone, sweet osmanthus hydrolat, rose hydrolat, antibacterial peptide and the like to develop a novel product for preventing and treating vulvovaginitis. The composition not only can resist bacteria synergistically, but also has small influence on lactobacillus, does not cause flora imbalance, and can effectively delay the generation of antibiotic resistance of bacteria.

Description

Technical Field

[0001] The present invention relates to an antibacterial composition, in particular to a composition with olive leaf extract as the antibacterial main component, a preparation method and application thereof, and belongs to the technical field of separating and purifying antibacterial active components of natural product extracts by using amino magnetic microspheres. Background Art

[0002] Gynecological diseases are one of the common health problems for women, among which infectious diseases are particularly common. Vaginitis is a common gynecological disease and is a frequently occurring disease. It is mainly caused by the destruction of the natural defense function of the vagina and the invasion of pathogens. It is mainly manifested by increased vaginal discharge, accompanied by clinical symptoms such as vulvar itching and odor. The incidence of vaginitis increases year by year, and has a high recurrence rate, which affects the patient's psychology, work and life.

[0003] At present, most of the drugs for treating various types of vaginitis are Western medicines that are antibacterial and antifungal. Long-term use can easily lead to drug dependence, and can also damage the internal environment and pH of the vagina itself, leading to dysbacteriosis and causing a series of serious complications, and easily leading to serious drug resistance, affecting further treatment. Natural products are the source of innovative drug discovery, with the advantages of multi-target synergistic treatment and low side effects. Modern research shows that olive leaves have strong antibacterial, anti-inflammatory, and antioxidant activities. And olive leaves, as waste after olive fruit picking, are low-cost, abundant renewable resources, which are conducive to realizing waste utilization and protecting the environment. Therefore, it is of great practical significance to develop an antibacterial compound drug with olive leaves as the main ingredient that can effectively alleviate the production of drug resistance.

[0004] In view of this situation, the present invention adopts a chemical co-precipitation method to prepare oleic acid-coated ferroferric oxide particles as a magnetic source, a microemulsion method to prepare magnetic polymer microspheres, and a surface chemical coating modification method to further amino-modify the magnetic microspheres after coating the surface of the magnetic microspheres with silicon dioxide. The obtained amino-modified magnetic microspheres are used for the separation and purification of antibacterial active components of natural product extracts (mainly olive leaf extract, and two or more of Sophora flavescens, Phellodendron amurense, Cnidium monnieri, Licorice, Radix Isatidis, Flos Lonicerae and Aloe extracts as auxiliary). After desorption, the product is concentrated by rotary evaporation and vacuum dried, and then the above active ingredients are compounded with a mixture of two or more monomer components such as niacinamide, coenzyme Q10, nootkatone, osmanthus hydrosol, rose hydrosol, antimicrobial peptides, etc. according to a formula to develop a new product for the prevention and treatment of vulvovaginitis. The composition not only has a significant bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans, but has little effect on Lactobacillus and will not cause bacterial imbalance. In addition, the addition of antimicrobial peptides can effectively delay the development of bacterial resistance to antibiotics, thereby providing a composition option with therapeutic effects for the prevention and treatment of vaginitis. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an antibacterial composition containing olive leaf extract, a preparation method and an application thereof in view of the deficiencies in the prior art. The olive leaf extract is mainly used, and is compounded with other ingredients after separation and purification by amino magnetic microspheres, especially the addition of independently designed antimicrobial peptides, which has a significant antibacterial effect and can effectively alleviate bacterial resistance.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: The present invention first provides a method for preparing an antibacterial composition containing olive leaf extract, which is mainly composed of olive leaf extract, which is separated and purified by amino magnetic microspheres and then compounded with other ingredients, comprising the following steps: (1) Preparation of amino magnetic microspheres: First, ferric chloride hexahydrate, ferrous sulfate, ammonia water and oleic acid are reacted to prepare oleic acid-coated ferrosoferric oxide particles, and then magnetic polymer microspheres are prepared by a microemulsion method. Then, the magnetic polymer microspheres are first coated with silicon dioxide on the surface of the magnetic microspheres through a hydrolysis and polycondensation reaction of ethyl orthosilicate, and then amino groups are further connected by introducing an amino silanol condensate. After Soxhlet extraction and vacuum drying, magnetic monodisperse porous microspheres are obtained, namely, the amino magnetic microspheres. (2) Preparation of natural product extracts: The natural products are olive leaf, sophora flavescens, phellodendron amurense, cnidium monnieri, liquorice, isatis root, honeysuckle and aloe vera. Different types of natural products are subjected to reflux extraction using ethanol as a solvent, and the extracts are combined after several reflux extractions. The different types of extracts are subjected to rotary evaporation, concentration and drying to obtain olive leaf extract, sophora flavescens extract, phellodendron amurense extract, cnidium monnieri extract, liquorice extract, isatis root extract, honeysuckle extract and aloe vera extract of certain concentrations. (3) Preparation of an antibacterial composition containing olive leaf extract: The olive leaf extract obtained in step (2) is mixed with any one, two or more of Sophora flavescens extract, Phellodendron amurense extract, Cnidium monnieri extract, Licorice extract, Radix Isatidis extract, Flos Lonicerae extract and Aloe extract according to the formula ratio and subjected to ultrasonic treatment to obtain an extract mixed solution; the amino magnetic microspheres obtained in step (1) are placed in the extract mixed solution for shaking magnetic adsorption, and the amino magnetic microspheres are used to adsorb the active ingredients in the extract, and after reaching adsorption equilibrium, the polymer is magnetically separated to obtain magnetic microspheres adsorbed with the active ingredients; the magnetic microspheres adsorbed with the active ingredients are placed in a desorption solvent for shaking desorption, and the adsorbed active ingredients are desorbed from the magnetic microspheres to obtain a desorption liquid, the desorption liquid is concentrated by rotary evaporation and then vacuum dried to obtain an extract, and finally mixed with an auxiliary agent, dissolved and ultrasonicated to obtain an antibacterial composition containing olive leaf extract.

[0007] In the above technical scheme, the method for preparing amino-modified magnetic microspheres in step (1) specifically comprises the following steps: ① Preparation of oleic acid-coated ferroferric oxide particles: Dispersing ferric chloride hexahydrate and ferrous sulfate in deionized water, heating under stirring conditions, then adding ammonia water and oleic acid to react, obtaining oleic acid-coated ferroferric oxide particles, using the oleic acid-coated ferroferric oxide particles as a magnetic source, then dispersing them in styrene, pouring them into a pear-shaped funnel, separating and washing them with deionized water, and obtaining a well-dispersed magnetic fluid; ② Preparation of magnetic polymer microspheres by microemulsion method: The magnetic fluid obtained in step ①, the hydrophobic monomer and the hydrophobic initiator are ultrasonically treated under the protection of ice to obtain an ultrasonic solution; at the same time, the emulsifier, the co-emulsifier and the deionized water are stirred in a three-necked flask, and after being fully stirred to form a uniform emulsion, they are added to the ultrasonic solution and prepolymerized at a certain temperature for a period of time, and then the reaction temperature is increased, and a hydrophilic initiator is added to the system and then polymerized for a period of time. After the reaction is completed, the magnetic balls are washed with distilled water magnetic separation to obtain magnetic polymer microspheres; ③ Coating silica on the surface of magnetic polymer microspheres: Add distilled water and ethanol to the magnetic polymer microspheres obtained in step ②, perform magnetic stirring under water bath conditions, add methacryloyloxyethyl trimethylammonium chloride to react after heating, cool after the reaction, add ammonia water and ethyl orthosilicate to the system to continue the reaction, stir and cool after the reaction, and use distilled water magnetic separation and washing to obtain magnetic polymer microspheres coated with silicon dioxide; ④ Connecting amino groups on the surface of magnetic polymer microspheres: The magnetic polymer microspheres coated with silica on the surface obtained in step ③ are dispersed in an ethanol solution, then mixed with ammonia water and stirred for a period of time, and then the amino silanol condensate is added. After stirring for a period of time, the temperature is raised to react. After the reaction is completed, distilled water is used for magnetic separation and cleaning, and then Soxhlet extraction and vacuum drying are performed to obtain magnetic monodisperse porous microspheres.

[0008] In the above technical scheme, in step ①, the mass ratio of oleic acid: ammonia water: ferrous sulfate: ferric chloride hexahydrate: deionized water is 1: (3-5): (3-5): (1-4): (5-8), and the mass ratio of oleic acid-coated ferrosoferric oxide particles to styrene is (2-5): (2-6); the temperature is increased under the stirring conditions, the speed during stirring is 100-200 rpm, preferably 150 rpm, and the temperature is increased to 60-90°C, preferably 80°C; the reaction time of the reaction is 2-4h, preferably 3h.

[0009] In the above technical scheme, in step ②, the hydrophobic monomers are methacrylic acid and divinylbenzene, and the volume ratio thereof is (10-30): (1-4); the hydrophobic initiator is benzoyl peroxide; the mass ratio of the magnetic fluid, the hydrophobic monomer, and the hydrophobic initiator is (10-30): (10-20): (5-10); the ultrasonic treatment is carried out under ice protection, and the conditions of the ultrasonic treatment are: the ultrasonic frequency is 40-120 KHZ, the ultrasonic time is 0.5-2 h, and the temperature during the ultrasonic process is 20-30°C.

[0010] In the above technical scheme, in step ②, the emulsifier is sodium dodecyl sulfate, the co-emulsifier is hexadecanol, the mass ratio of the emulsifier, the co-emulsifier and deionized water is (0.05-0.15): (0.4-0.6): (20-30); the mass ratio of the emulsion and the ultrasonic solution is (20-30): (3-10); the hydrophilic initiator is potassium persulfate, and the mass ratio of potassium persulfate to benzoyl peroxide is (3-7): (50-100).

[0011] In the above technical scheme, in step ②, the prepolymerization reaction has the following reaction conditions: prepolymerization at 50-65°C for 1-4 h, preferably prepolymerization at 60°C for 1-2 h; the further increasing of the reaction temperature refers to increasing the reaction temperature to 66-90°C, preferably increasing the temperature to 70°C; the polymerization reaction has a reaction time of 12-36 h, preferably adding a hydrophilic initiator and then performing the polymerization reaction for 24 h.

[0012] In the above technical scheme, in step ③, the mass ratio of the magnetic polymer microspheres, distilled water and ethanol is (0.02-0.1): (1-4): (8-14); the magnetic stirring under water bath conditions refers to magnetic stirring in a magnetic stirring water bath pot, the stirring speed is 100-200rpm, and the water bath temperature is set to 50-80°C, preferably 70°C; after reaching the set temperature, methacryloyloxyethyl trimethyl ammonium chloride is added to react, and the reaction time is 4-8 h. After the reaction is completed, the temperature is lowered to 45-55°C, preferably 50°C, and the mass ratio of methacryloyloxyethyl trimethyl ammonium chloride to the magnetic polymer microspheres is (1-3): (2-10); ammonia water and tetraethyl orthosilicate are added to the system to continue the reaction, and the volume ratio of ammonia water, tetraethyl orthosilicate and methacryloyloxyethyl trimethyl ammonium chloride is (20-40): (10-30): (10-40), and the mass concentration of ammonia water is 25%; the reaction time is 1-3 h, preferably 2 h, after the reaction is completed, stirring and cooling to 20-30°C.

[0013] In the above technical scheme, in step ④, the mass ratio of the magnetic polymer microspheres coated with silica on the surface to the ethanol solution is (0.02-0.1): (1-3), and the mass concentration of the ethanol solution is 60-99%, preferably 75%; the mass ratio of the magnetic polymer microspheres coated with silica on the surface to the ammonia water is (2-10): (10-20), and the mass concentration of the ammonia water is 20-40%, preferably 25%; the amino silanol condensate is aminopropyltrimethoxysilane or (3-aminopropyl)triethoxysilane, and the mass ratio of the magnetic polymer microspheres coated with silica on the surface to the amino silanol condensate is (20-100): (5-10); after adding the ammonia water, stir at room temperature for 0.2-1 h, preferably 0.5 h, and after adding the amino silanol condensate, stir continuously at 25-40°C for 1-4 h, preferably at 35°C for 3 h; the temperature-raising reaction refers to the reaction at 50-75°C for 0.5-2 h, preferably at 65°C for 1 h.

[0014] In the above technical scheme, in step (2), the olive leaf extract is specifically prepared by the following steps: weighing 20-40 g of shade-dried olive leaves, adding 8-14 times the amount of ethanol with a mass concentration of 45-80%, and reflux extraction at 40-50°C for 2 times, each time for 1-2.5 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original olive leaves).

[0015] In the above technical solution, in step (2), the specific preparation method of the Sophora flavescens extract comprises the following steps: weighing 5-10 g of Sophora flavescens powder, adding 8-12 times the amount of 70-95% ethanol by mass concentration, and reflux extraction at 40-50°C for 2 times, each time for 2-3 hours; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original Sophora flavescens powder).

[0016] In the above technical scheme, in step (2), the specific preparation method of the Phellodendron amurense extract comprises the following steps: weighing 5-10 g of Phellodendron amurense slices, adding 8-12 times the amount of 60-95% ethanol by mass concentration, and reflux extraction at 40-50°C for 3 times, each time for 1-2.5 hours; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original Phellodendron amurense slices).

[0017] In the above technical scheme, in step (2), the Cnidium monnieri extract is specifically prepared by the following steps: weighing 5-10 g of Cnidium monnieri, adding 8-12 times the amount of 45-75% ethanol by mass, and reflux extracting at 40-50°C for 2 times, each time for 1-2.5 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original Cnidium monnieri).

[0018] In the above technical solution, in step (2), the licorice extract is specifically prepared by the following steps: weighing 5-10 g of licorice, adding 8-12 times the amount of 45-75% ethanol by mass, and reflux extracting at 40-50°C for 2 times, each time for 1-2.5 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original licorice).

[0019] In the above technical scheme, in step (2), the specific preparation method of the Radix Isatidis extract comprises the following steps: weighing 5-10 g of Radix Isatidis, adding 8-12 times the amount of 45-75% ethanol by mass, and reflux extraction at 40-50°C for 2 times, each time for 1-2.5 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original Radix Isatidis).

[0020] In the above technical scheme, in step (2), the specific preparation method of the honeysuckle extract comprises the following steps: weighing 5-10 g of honeysuckle, adding 6-10 times the amount of 45-75% ethanol by mass concentration, and reflux extraction at 40-50°C for 2 times, each time for 1-2 hours; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original honeysuckle).

[0021] In the above technical solution, in step (2), the aloe extract is specifically prepared by the following steps: weighing 5-10 g of aloe, adding 8-12 times the amount of ethanol with a mass concentration of 60-75%, and reflux extraction at 40-50°C for 2 times, each time for 1-2 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL (based on the mass of the original aloe).

[0022] In the above technical scheme, in step (3), the olive leaf extract is mixed with any one, two or more of Sophora flavescens extract, Phellodendron amurense extract, Cnidium monnieri extract, Licorice extract, Isatis indigotica extract, Honeysuckle extract and Aloe vera extract according to the formula ratio and then ultrasonically obtained to obtain an extract mixed solution, and the conditions for ultrasonic mixing are: the ultrasonic frequency is 40-120 KHZ, the ultrasonic time is 0.5-2 h, and the temperature during the ultrasonic process is 20-30 ° C.

[0023] In the above technical scheme, in step (3), during the magnetic adsorption and desorption process, the mixed extract solution is placed in a centrifuge tube, and amino magnetic microspheres are added at the same time, and the solution is continuously shaken in a constant temperature oscillator for magnetic adsorption. The mass ratio of the total mass of the natural products (i.e., original olive leaves, sophora flavescens, phellodendron amurense, cnidium monnieri, licorice, isatis root, honeysuckle and aloe) in the mixed extract solution to the amino magnetic microspheres is (7-14): (2-6), and the conditions for magnetic adsorption are: adsorption temperature 20-35°C, adsorption speed 110-150 rpm, and adsorption time 4-8 h; then, an ethanol solution with a mass concentration of 50-75% is used as a desorption solvent, and the solution is continuously shaken in a constant temperature oscillator for desorption. The mass ratio of the desorption solvent to the amino magnetic microspheres is (200-500): (2-6), and the conditions for desorption are: adsorption temperature 20-35°C, adsorption speed 110-150 rpm, and adsorption time 4-8 h; the rotary evaporation of the desorption liquid refers to rotary evaporation of the desorption liquid until there is no alcohol taste, and then vacuum drying at 50°C to obtain the extract.

[0024] In the above technical scheme, in step (3), the auxiliary agent is any one, two or more of nicotinamide, coenzyme Q10, nootkatone, osmanthus hydrosol, rose hydrosol, and antimicrobial peptides; the antimicrobial peptides mainly act on bacterial cell membranes and are not easy to develop drug resistance, and their amino acid sequence is: (RW)n PPPP (KW)n, n= (2-4); the extract and the auxiliary agent are mixed and dissolved by ultrasonication, and the ultrasonic conditions are: the ultrasonic frequency is 40-120 KHZ, the ultrasonic time is 0.5-1 h, and the temperature during the ultrasonic process is 20-30 °C.

[0025] In the above technical solution, the antibacterial composition containing olive leaf extract includes the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice root: Isatis indigotica: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokatone: Osmanthus hydrosol: Rose hydrosol, antimicrobial peptides = (35-60): (0-20): (0-20): (0-20): (0-20): (0-20): (0-20): (0-20): (0-10): (0-10): (0-10): (0-10): (0-10): (0-6); among which, the concentration of natural product extracts is based on the mass of the natural product initially weighed, and its total weight proportion is not less than 85%. Niacinamide, coenzyme Q10, Nokatone, osmanthus hydrosol, rose hydrosol and antimicrobial peptides are auxiliary ingredients, and the sum of their total weight proportions is not higher than 15%.

[0026] In the above technical solution, the antibacterial composition containing olive leaf extract preferably includes the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice: Isatis root: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokadone: Osmanthus hydrosol: Rose hydrosol: Antibacterial peptide = (40-55): (0-16): (0-16): (0-16): (0-16): (0-16): (0-16): (0-16): (0-6): (0-6): (0-6): (0-6): (0-6): (0-2).

[0027] In the above technical solution, the antibacterial composition containing olive leaf extract further preferably includes the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice: Isatis root: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokadone: Osmanthus hydrosol: Rose hydrosol: Antimicrobial peptide = (40-55): (0-16): (0.01-16): (0.01-16): (0-16): (0.01-16): (0-16): (0.01-16): (0.001-6): (0-6): (0-6): (0.001-6): (0-6): (0.001-2).

[0028] In the above technical solution, the antibacterial composition containing olive leaf extract further preferably includes the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice: Isatis root: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokadone: Osmanthus hydrosol: Rose hydrosol: Antimicrobial peptide = (40-55): (0-16): (0.01-16): (0-16): (0-16): (0-16): (0-16): (0.01-16): (0-6): (0.001-6): (0-6): (0.001-6): (0-6): (0.001-2).

[0029] In the above technical solution, the antibacterial composition containing olive leaf extract further preferably includes the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice: Isatis root: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokadone: Osmanthus hydrosol: Rose hydrosol: Antimicrobial peptide = (40-55): (0.01-16): (0-16): (0.01-16): (0-16): (0.01-16): (0-16): (0-6): (0-6): (0.001-6): (0.001-6): (0-6): (0.001-2).

[0030] The present invention also provides an antibacterial composition containing olive leaf extract prepared by the above method, wherein the composition is in any pharmaceutical dosage form, such as tablets, capsules, suppositories, gels, ointments, etc.

[0031] The present invention also provides an application of the antibacterial composition containing olive leaf extract in preparing medicine for preventing and treating vulvovaginitis.

[0032] The present invention has the following advantages over the prior art: 1. The preparation process of the aminated magnetic microspheres of the present invention is simple and clear, easy to operate, and enhances the adsorption force of the magnetic microspheres for antibacterial active ingredients. When separating and purifying antibacterial active ingredients in a mixed solution mainly containing olive leaves and supplemented by two or more extracts of sophora flavescens, phellodendron, cnidium monnieri, liquorice, isatis root, honeysuckle, and aloe, the content of oleuropein reaches more than 40%, and the content of luteolin reaches more than 35%. The modified magnetic microspheres prepared by the present invention can be quickly separated under an external magnetic field and can be regenerated and reused.

[0033] 2. The preparation process of the olive leaf extract of the present invention is simple, the yield of oleuropein reaches more than 85%, and the content reaches more than 35%. Moreover, the waste of olive leaves can be reused, which has good ecological, economic and social benefits.

[0034] 3. The compound of the present invention is mainly composed of olive leaf extract, and is a new product for preventing and treating vulvovaginitis. The composition has a significant bactericidal effect on Escherichia coli, Staphylococcus aureus, and Candida albicans, and has little effect on Lactobacillus, and has the advantage of selective bactericidal effect. Moreover, the addition of new antimicrobial peptides can destroy the cell membrane and effectively delay the development of bacterial resistance to antibiotics, providing a composition with therapeutic effect for the prevention and treatment of vaginitis. DETAILED DESCRIPTION

[0035] The specific implementation of the technical solution of the present invention is described in detail below, but the present invention is not limited to the following description. The present invention is described in detail below through specific embodiments: Example 1: An antibacterial composition containing olive leaf extract The antibacterial composition containing olive leaf extract is prepared by the following preparation method: (1) Preparation of amino magnetic microspheres: ① Preparation of oleic acid-coated ferroferric oxide particles: 25 g of ferric chloride hexahydrate and 15 g of ferrous sulfate were dispersed in 60 mL of deionized water, and the temperature was raised under stirring at a stirring speed of 150 rpm. When the temperature reached 80°C, 40 mL of ammonia water and 10 mL of oleic acid were quickly added. After reacting for 3 h, the collected products were subjected to magnetic separation and washing to obtain oleic acid-coated ferrosoferric oxide nanoparticles; 25 g of ferrosoferric oxide nanoparticles were dispersed in 25 mL of styrene, poured into a pear-shaped funnel, separated and washed with deionized water, and a well-dispersed magnetic fluid was obtained; ② Preparation of magnetic polymer microspheres by microemulsion method: Take 2 mL of the ferroferric oxide-styrene magnetic fluid prepared in step ①, mix it with 0.25 mL of divinylbenzene (DVB), 1.5 mL of methyl methacrylate (MAA) and 0.75 g of benzoyl peroxide (BPO), and ultrasonicate it under the protection of ice cubes to avoid the temperature rising during the ultrasonic process and inducing the self-polymerization of styrene. The conditions of ultrasonic treatment are: the ultrasonic frequency is 120MHZ, the ultrasonic time is 1h, and the temperature during the ultrasonic process is 30℃. At the same time, stir 0.1g of sodium dodecyl sulfate (SDS) (emulsifier), 0.53g of hexadecanol (CA) (co-emulsifier) ​​and 25 mL of deionized water in a 100 mL three-necked flask. After it is fully stirred to form a uniform emulsion, add the previously ultrasonicated solution. After prepolymerizing the above mixture at 60℃ for 2 h, the temperature is raised to 70℃, 0.05g of hydrophilic initiator potassium persulfate (KPS) is added, and the polymerization is carried out for 24 h. The magnetic polymer microspheres are obtained after magnetic separation and washing of the magnetic spheres with distilled water.

[0036] ③ Coating silica on the surface of magnetic polymer microspheres: Take 0.05g of the magnetic polymer microspheres prepared in step ②, add 2 mL of distilled water and 10 mL of ethanol, stir in a magnetic stirring water bath, set the temperature to 70°C, and stir at 150 rpm. When the temperature rises to 70°C, add 25 µL of methacryloyloxyethyl trimethylammonium chloride (MTC), react for 6 h, then cool to 50°C, add 30 µL of 25% ammonia water and 20 µL of ethyl orthosilicate, react for two hours, stir and cool to 30°C, and wash with distilled water magnetic separation to obtain magnetic polymer microspheres coated with silica on the surface.

[0037] ④ Connecting amino groups on the surface of magnetic polymer microspheres: Take 0.05 g of magnetic spheres from step ③ and disperse them in 2.5 mL of 75% ethanol solution, then mix with 12.5 μL of 25% ammonia water and stir at room temperature for 0.5 h. Then add 6.25 μL of (3-aminopropyl) triethoxysilane (APTES), stir continuously at 35°C for 3 h, then heat to 65°C for 1 h, and wash by distilled water magnetic separation. After Soxhlet extraction, vacuum drying was performed to obtain magnetic monodisperse porous microspheres.

[0038] (2) Preparation of natural product extracts: ① Preparation of olive leaf extract: Weigh 10 g of shade-dried olive leaves, use 12 / 10 times the amount (reflux extraction twice, the first time is 12 times the amount, the second time is 10 times the amount) of 75% ethanol as solvent, reflux extraction twice at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for later use.

[0039] ② Preparation of Phellodendron chinense extract: Weigh 10 g of Phellodendron amurense slices, use 10 / 12 / 10 times volume (reflux extraction 3 times, the first time is 10 times volume, the second time is 12 times volume, and the third time is 10 times volume) of 75% ethanol as solvent, reflux extraction 3 times at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for use.

[0040] ③Preparation of Cnidium monnieri extract: Weigh 10 g of Cnidium monnieri, use 10 / 12 times ((reflux extraction twice, the first time is 10 times the amount, the second time is 12 times the amount)) 60% ethanol as solvent, reflux extraction twice at 45°C, each time for 1.5 h. Combine the extracts, and concentrate them by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for later use.

[0041] ④ Preparation of Radix Isatidis Extract: Weigh 10 g of Radix Isatidis, add 10 / 12 times (reflux extraction twice, 10 times for the first time and 12 times for the second time) 75% ethanol as solvent, reflux extraction twice at 45°C, 1.5 h each time. Combine the extracts, concentrate to 100 mL by rotary evaporation at 40-50°C, i.e., the final concentration is 0.1 g / mL for later use.

[0042] ⑤Preparation of aloe extract: Weigh 10 g of aloe vera, add 8 / 10 times (reflux extraction twice, 8 times the first time, 10 times the second time) 75% ethanol as solvent, reflux extraction twice at 45°C, 1.5 h each time. Combine the extracts, concentrate to 100 mL by rotary evaporation at 40-50°C, that is, the final concentration is 0.1 g / mL for later use.

[0043] (3) Preparation of an antibacterial composition containing olive leaf extract: Take 40 mL of olive leaf extract, 12 mL of Phellodendron chinense extract, 12 mL of Cnidium monnieri extract, 12 mL of Radix Isatidis extract, and 12 mL of Aloe extract prepared in step (2), dissolve them in 250 mL of deionized water, and mix and sonicate for 30 min (25°C, 120 MHZ) to obtain a mixed extract solution. Accurately weigh 5 g of the amino magnetic microspheres obtained in step (1) and place them in a 500 mL stoppered conical flask, add the above mixed extract solution, and continuously shake and adsorb for 6 h (25°C, 125 rpm) in a constant temperature oscillator. After reaching adsorption equilibrium, magnetically separate the polymer. Determine the concentration of the adsorbed residual liquid by liquid chromatography, and calculate the adsorption capacity of the material for the above extracts. A 70% ethanol solution was used as a desorption solvent, 250 mL was taken and added to the amino magnetic microspheres adsorbed with the active ingredient, and the mixture was continuously shaken in a constant temperature oscillator for desorption for 6 h (25°C, 125 rpm), the adsorbed active ingredient was desorbed from the amino magnetic microspheres, the polymer was magnetically separated, the desorption liquid obtained after desorption was concentrated by rotary evaporation, and then vacuum dried at 50°C to obtain an extract, followed by adding 0.6 g niacinamide, 0.58 g osmanthus hydrosol, and 0.02 g antimicrobial peptide, and the mixture was dissolved and ultrasonicated for 30 min (25°C, 120 MHZ) to obtain a compound antibacterial product with olive leaf as the main component, which is the antibacterial composition containing olive leaf extract; wherein the amino acid sequence of the antimicrobial peptide is: RWRWRWPPPPKWKWKW.

[0044] Example 2: An antibacterial composition containing olive leaf extract The antibacterial composition containing olive leaf extract is prepared by the following preparation method: (1) Preparation of amino magnetic microspheres: ① Preparation of oleic acid-coated ferroferric oxide particles: 25 g of ferric chloride hexahydrate and 15 g of ferrous sulfate were dispersed in 60 mL of deionized water, and the temperature was raised under stirring at a stirring speed of 150 rpm. When the temperature reached 80°C, 40 mL of ammonia water and 10 mL of oleic acid were quickly added. After reacting for 3 h, the collected products were subjected to magnetic separation and washing to obtain oleic acid-coated ferrosoferric oxide nanoparticles; 25 g of ferrosoferric oxide nanoparticles were dispersed in 25 mL of styrene, poured into a pear-shaped funnel, separated and washed with deionized water, and a well-dispersed magnetic fluid was obtained; ② Preparation of magnetic polymer microspheres by microemulsion method: Take 2 mL of the ferroferric oxide-styrene magnetic fluid prepared in step ①, mix it with 0.25 mL of divinylbenzene (DVB), 1.5 mL of methyl methacrylate (MAA) and 0.75 g of benzoyl peroxide (BPO), and ultrasonicate it under the protection of ice cubes to avoid the temperature rising during the ultrasonic process and inducing the self-polymerization of styrene. The conditions of ultrasonic treatment are: the ultrasonic frequency is 120MHZ, the ultrasonic time is 1h, and the temperature during the ultrasonic process is 30℃. At the same time, stir 0.1g of sodium dodecyl sulfate (SDS) (emulsifier), 0.53g of hexadecanol (CA) (co-emulsifier) ​​and 25 mL of deionized water in a 100 mL three-necked flask. After it is fully stirred to form a uniform emulsion, add the previously ultrasonicated solution. After prepolymerizing the above mixture at 60℃ for 2 h, the temperature is raised to 70℃, 0.05g of hydrophilic initiator potassium persulfate (KPS) is added, and the polymerization is carried out for 24 h. The magnetic polymer microspheres are obtained after magnetic separation and washing of the magnetic spheres with distilled water.

[0045] ③ Coating silica on the surface of magnetic polymer microspheres: Take 0.05g of the magnetic polymer microspheres prepared in step ②, add 2 mL of distilled water and 10 mL of ethanol, stir in a magnetic stirring water bath, set the temperature to 70°C, and stir at 150 rpm. When the temperature rises to 70°C, add 25 µL of methacryloyloxyethyl trimethylammonium chloride (MTC), react for 6 h, then cool to 50°C, add 30 µL of 25% ammonia water and 20 µL of ethyl orthosilicate, react for two hours, stir and cool to 30°C, and wash with distilled water magnetic separation to obtain magnetic polymer microspheres coated with silica on the surface.

[0046] ④ Connecting amino groups on the surface of magnetic polymer microspheres: Take 0.05 g of magnetic spheres from step ③ and disperse them in 2.5 mL of 75% ethanol solution, then mix with 12.5 μL of 25% ammonia water and stir at room temperature for 0.5 h. Then add 6.25 μL of (3-aminopropyl) triethoxysilane (APTES), stir continuously at 35°C for 3 h, then heat to 65°C for 1 h, and wash by distilled water magnetic separation. After Soxhlet extraction, vacuum drying was performed to obtain magnetic monodisperse porous microspheres.

[0047] (2) Preparation of natural product extracts: ① Preparation of olive leaf extract: Weigh 30 g of shade-dried olive leaves, use 12 / 10 times the amount (reflux extraction twice, the first time is 12 times the amount, the second time is 10 times the amount) of 75% ethanol as solvent, reflux extraction twice at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for later use.

[0048] ② Preparation of Phellodendron chinense extract: Weigh 10 g of Phellodendron amurense slices, use 10 / 12 / 10 times volume (reflux extraction 3 times, the first time is 10 times volume, the second time is 12 times volume, and the third time is 10 times volume) of 75% ethanol as solvent, reflux extraction 3 times at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for use.

[0049] ③Preparation of aloe extract: Weigh 10 g of aloe vera, add 8 / 10 times (reflux extraction twice, 8 times the first time, 10 times the second time) 75% ethanol as solvent, reflux extraction twice at 45°C, 1.5 h each time. Combine the extracts, concentrate to 100 mL by rotary evaporation at 40-50°C, that is, the final concentration is 0.1 g / mL for later use.

[0050] (3) Preparation of an antibacterial composition containing olive leaf extract: Take 55 mL of olive leaf extract, 15 mL of Phellodendron chinense extract, and 16 mL of aloe extract prepared in step (2), dissolve them in 250 mL of deionized water, mix and sonicate for 30 min (25°C, 120 MHZ) to obtain a mixed extract solution. Accurately weigh 4 g of the amino magnetic microspheres obtained in step (1) and place them in a 500 mL stoppered conical flask, add the above mixed extract solution, and continuously shake and adsorb for 6 h (25°C, 125 rpm) in a constant temperature oscillator. After reaching adsorption equilibrium, magnetically separate the polymer. Determine the concentration of the adsorbed residual liquid by liquid chromatography, and calculate the adsorption capacity of the material for the above extracts. A 70% ethanol solution was used as a desorption solvent, 250 mL was taken and added to the amino magnetic microspheres adsorbed with the active ingredient, and the mixture was continuously shaken in a constant temperature oscillator for desorption for 6 h (25°C, 125 rpm), the adsorbed active ingredient was desorbed from the amino magnetic microspheres, the polymer was magnetically separated, the desorption liquid obtained after desorption was concentrated by rotary evaporation, and vacuum dried at 50°C to obtain an extract, followed by adding 0.67 g of coenzyme Q10, 0.7 g of osmanthus hydrosol, and 0.03 g of antimicrobial peptide, and the mixture was dissolved and ultrasonicated for 30 min (25°C, 120 MHZ) to obtain a compound antibacterial product with olive leaf as the main component, which is the antibacterial composition containing olive leaf extract; wherein the amino acid sequence of the antimicrobial peptide is: RWRWRWPPPPKWKWKW.

[0051] Example 3: An antibacterial composition containing olive leaf extract The antibacterial composition containing olive leaf extract is prepared by the following preparation method: (1) Preparation of amino magnetic microspheres: ① Preparation of oleic acid-coated ferroferric oxide particles: 25 g of ferric chloride hexahydrate and 15 g of ferrous sulfate were dispersed in 60 mL of deionized water, and the temperature was raised under stirring at a stirring speed of 150 rpm. When the temperature reached 80°C, 40 mL of ammonia water and 10 mL of oleic acid were quickly added. After reacting for 3 h, the collected products were subjected to magnetic separation and washing to obtain oleic acid-coated ferrosoferric oxide nanoparticles; 25 g of ferrosoferric oxide nanoparticles were dispersed in 25 mL of styrene, poured into a pear-shaped funnel, separated and washed with deionized water, and a well-dispersed magnetic fluid was obtained; ② Preparation of magnetic polymer microspheres by microemulsion method: Take 2 mL of the ferroferric oxide-styrene magnetic fluid prepared in step ①, mix it with 0.25 mL of divinylbenzene (DVB), 1.5 mL of methyl methacrylate (MAA) and 0.75 g of benzoyl peroxide (BPO), and ultrasonicate it under the protection of ice cubes to avoid the temperature rising during the ultrasonic process and inducing the self-polymerization of styrene. The conditions of ultrasonic treatment are: the ultrasonic frequency is 120MHZ, the ultrasonic time is 1h, and the temperature during the ultrasonic process is 30℃. At the same time, stir 0.1g of sodium dodecyl sulfate (SDS) (emulsifier), 0.53g of hexadecanol (CA) (co-emulsifier) ​​and 25 mL of deionized water in a 100 mL three-necked flask. After it is fully stirred to form a uniform emulsion, add the previously ultrasonicated solution. After prepolymerizing the above mixture at 60℃ for 2 h, the temperature is raised to 70℃, 0.05g of hydrophilic initiator potassium persulfate (KPS) is added, and the polymerization is carried out for 24 h. The magnetic polymer microspheres are obtained after magnetic separation and washing of the magnetic spheres with distilled water.

[0052] ③ Coating silica on the surface of magnetic polymer microspheres: Take 0.05g of the magnetic polymer microspheres prepared in step ②, add 2 mL of distilled water and 10 mL of ethanol, stir in a magnetic stirring water bath, set the temperature to 70°C, and stir at 150 rpm. When the temperature rises to 70°C, add 25 µL of methacryloyloxyethyl trimethylammonium chloride (MTC), react for 6 h, then cool to 50°C, add 30 µL of 25% ammonia water and 20 µL of ethyl orthosilicate, react for two hours, stir and cool to 30°C, and wash with distilled water magnetic separation to obtain magnetic polymer microspheres coated with silica on the surface.

[0053] ④ Connecting amino groups on the surface of magnetic polymer microspheres: Take 0.05 g of magnetic spheres from step ③ and disperse them in 2.5 mL of 75% ethanol solution, then mix with 12.5 μL of 25% ammonia water and stir at room temperature for 0.5 h. Then add 6.25 μL of (3-aminopropyl) triethoxysilane (APTES), stir continuously at 35°C for 3 h, then heat to 65°C for 1 h, and wash with distilled water by magnetic separation. After Soxhlet extraction, vacuum drying is performed to obtain magnetic monodisperse porous microspheres.

[0054] (2) Preparation of natural product extracts: ① Preparation of olive leaf extract: Weigh 30 g of shade-dried olive leaves, use 12 / 10 times the amount (reflux extraction twice, the first time is 12 times the amount, the second time is 10 times the amount) of 75% ethanol as solvent, reflux extraction twice at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for later use.

[0055] ② Preparation of Sophora flavescens extract: Weigh 10 g of Sophora flavescens powder, use 8 / 12 times volume (reflux extraction twice, the first time is 8 times volume, the second time is 12 times volume) of 95% ethanol as solvent, reflux extraction twice at 45°C, each reflux extraction for 2 h, combine the extracts, and concentrate them by rotary evaporation at 40-50°C to 100 mL, that is, the final concentration is 0.1 g / mL for later use.

[0056] ③Preparation of Cnidium monnieri extract: Weigh 10 g of Cnidium monnieri, use 10 / 12 times (reflux extraction twice, 10 times for the first time and 12 times for the second time) 60% ethanol as solvent, reflux extraction twice at 45°C, 1.5 h each time. Combine the extracts, concentrate to 100 mL by rotary evaporation at 40-50°C, i.e., the final concentration is 0.1 g / mL for later use.

[0057] ④Preparation of honeysuckle extract: Weigh 10 g of honeysuckle, add 8 / 10 times (reflux extraction twice, 8 times for the first time, 10 times for the second time) 75% ethanol as solvent, reflux extraction twice at 45°C, 1.5 h each time. Combine the extracts, evaporation concentrate to 100 mL at 40-50°C, i.e., the final concentration is 0.1 g / mL for later use.

[0058] (3) Preparation of an antibacterial composition containing olive leaf extract: 46 mL of olive leaf extract, 13 mL of Sophora flavescens extract, 14 mL of honeysuckle extract, and 15 mL of Cnidium monnieri extract prepared in step (2) were dissolved in 250 mL of deionized water and mixed by ultrasonication for 30 min ((25°C, 120 MHZ)) to obtain a mixed extract solution. Accurately weigh 5 g of the amino-modified magnetic microspheres obtained in step (1) and place them in a 500 mL stoppered conical flask, and add the above mixed extract solution, and continuously shake and adsorb for 6 h in a constant temperature oscillator (25°C, 125 rpm). After reaching adsorption equilibrium, the polymer was magnetically separated. The concentration of the adsorbed residual liquid was determined by liquid chromatography, and the adsorption capacity of the material for the above extracts was calculated. A 70% ethanol solution was used as a desorption solvent, 250 mL was taken and added to the amino magnetic microspheres adsorbed with the active ingredient, and the mixture was continuously shaken in a constant temperature oscillator for desorption for 6 h (25°C, 125 rpm), the adsorbed active ingredient was desorbed from the amino magnetic microspheres, the polymer was magnetically separated, the desorption liquid obtained after desorption was concentrated by rotary evaporation, and vacuum dried at 50°C to obtain an extract, followed by adding 0.60 g of nocardone, 0.56 g of osmanthus hydrosol, and 0.04 g of antimicrobial peptide, and the mixture was dissolved and ultrasonicated for 30 min (25°C, 120 MHZ) to obtain a compound antibacterial product with olive leaf as the main component, which is the antibacterial composition containing olive leaf extract; wherein the amino acid sequence of the antimicrobial peptide is: RWRWRWPPPPKWKWKW.

[0059] Application Example 1: Antibacterial effect of an antibacterial composition containing olive leaf extract The amino acid sequence of the antimicrobial peptide in Application Example 1 is (RW)n PPPP (KW)n, which is designed by taking into account factors such as net charge, hydrophobicity, amphipathicity, and peptide helical structure, and synthesized by Hangzhou Gutuo Biotechnology Co., Ltd.

[0060] The antibacterial effect experiment of the antibacterial composition containing olive leaf extract prepared in Example 1 includes the following steps.

[0061] 1. Inoculate the experimental bacteria (Escherichia coli, Staphylococcus aureus, Candida albicans, Lactobacillus) into tryptic soy bean broth (TSB), shake at 25 °C for 12 h, activate twice, and set aside.

[0062] 2. Optimization of peptide amino acid sequence The minimum inhibitory concentration (MIC) of antimicrobial peptides with different amino acid sequences was determined by the test tube double dilution method. The results in Table 1 show that the antibacterial efficacy of the peptide increases with the increase of repeating units, and further increasing the chain length will lead to a decrease or even loss of antibacterial activity. N = (2-4) has a good antibacterial effect, and n = 3 has the best antibacterial effect. The peptide sequence with n = 3 was selected for subsequent experiments (i.e.) RWRWRWPPPPKWKWKW.

[0063] Table 1 MICs of antimicrobial peptide compound drugs with different amino acid sequences

[0064] 3. MIC and MBC of olive leaf extract antibacterial compound drugs against bacteria The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the molecular compound were determined by the test tube double dilution method, and the turbidity of each test tube was observed and the results were recorded. MIC is the olive leaf concentration at the maximum dilution multiple at which the liquid in the test tube appears clear and transparent. Then the MIC concentration and the olive extract samples of the two adjacent dilutions before and after it were spread on the ordinary agar medium. MBC is the test tube concentration at which no more than 5 colonies are formed in the ordinary agar medium. Table 2 shows that the olive leaf extract antibacterial compound drug has a good antibacterial effect on Escherichia coli, Staphylococcus aureus, and Candida albicans, has little effect on lactobacilli, and will not cause an imbalance in the flora.

[0065] Table 2 MIC and MBC of the antibacterial compound drug of olive leaf extract Group MIC (µg / mL) MBC (µg / mL) Escherichia coli 0.030 0.041 Staphylococcus aureus 0.021 0.029 Candida albicans 0.026 0.034 Lactobacilli - - 4. Antibacterial rate of olive leaf extract antibacterial compound medicine Take 0.5 mL of freshly prepared bacterial suspension and drop it into 4.5 mL of sample solution (concentration is 0.4%). After mixing, start timing for 5 minutes. Use a quantitative pipette to draw 0.5 mL of bacterial drug suspension into a 4.5 mL test tube containing PBS, mix thoroughly, neutralize for 10 minutes, and dilute 10 times in series. Select the appropriate dilution, draw 1.0 mL and inoculate the plate respectively. Inoculate 2 plates per tube. Pour the nutrient agar medium melted at 40 ℃ ~ 45 ℃ into the plate added with the sample solution, 15mL ~ 20mL per plate, rotate the plate to mix thoroughly, turn the plate over after the agar solidifies, and culture at 36 ℃ ± 1 ℃ for 48 hours to count the number of live bacteria. At the same time, the diluent is used instead of the sample to carry out parallel experiments as a negative control; 0.4% tetracycline hydrochloride is used instead of the sample to carry out parallel experiments as a positive control. Table 3 shows that the antibacterial compound drug of olive leaf extract has strong antibacterial properties against Escherichia coli, Staphylococcus aureus and Candida albicans, has no effect on lactobacilli, and will not cause bacterial flora imbalance.

[0066] Table 3 Antibacterial rate of olive leaf extract antibacterial compound medicine

[0067] 5. Inhibition zone of olive leaf extract antibacterial compound drug Evenly spread the bacterial suspension (100 μL, 1×10 7 CFU / mL), and then placed the Oxford cup on the culture medium, added 250 μL of olive leaf extract antibacterial compound drugs to each Oxford, and measured the inhibition zone diameter (IZD) after culturing at 37 ℃ for 24 h. Among them, 0.4% tetracycline hydrochloride was used as a positive control, and the sterile saline group was used as a negative control. The results showed that the olive leaf extract antibacterial compound drug had strong antibacterial properties against Escherichia coli, Staphylococcus aureus, and Candida albicans, had no effect on lactobacilli, and would not cause bacterial imbalance.

[0068] Table 4 Inhibition zone of the antibacterial compound drug of olive leaf extract

[0069] 6. Effects of olive leaf extract antibacterial compound on inflammatory factors in RAW 264.7 cells infected with Escherichia coli / Staphylococcus aureus / Candida albicans RAW 264.7 cells were revived and DMEM containing 10% FBS was added and cultured at 37°C and 5% CO2. RAW264.7 cells in the logarithmic growth phase were taken and plated at 4×10 cells per well. 5 The cells were inoculated in a 24-well plate at a density of 10 cells / well. After 24 hours, the cells were infected with Escherichia coli / Staphylococcus aureus / Candida albicans (MOI = 20), and the antibacterial compound drug of olive leaf extract (experimental group), 0.4% tetracycline hydrochloride (positive control group) or an equal volume of sterile saline was added as the negative control group. Each group had 3 replicates, and the uninfected cell group was set as the blank group. After 8 hours of infection, the cell supernatant was collected, and the IL-1β and IL-6 levels in the cell supernatant were detected by ELISA.

[0070] Table 5 Effects of olive leaf extract antibacterial compound drugs on cellular inflammatory factors Group IL-1β (ng / L) IL-6 (ng / L) Blank Group 80.12 ± 6.13 84.54 ± 6.71 Negative control group 102.29 ± 5.82 114.76 ± 8.17 Positive control group 86.36 ± 8.12 97.33 ± 5.79 Experimental Group 89.72 ± 7.36 99.74 ± 5.34 The above results indicate that the antibacterial compound medicine of olive leaf extract can alleviate the cellular inflammatory response caused by the above bacterial and fungal infections.

[0071] 7. Olive leaf extract antibacterial compound drug delays the development of bacterial resistance to antibiotics To verify whether the combination of drugs can delay the development of resistance of Staphylococcus aureus to multiple types of antibiotics. Taking Staphylococcus aureus as an example, first, the minimum inhibitory concentration (MIC) of multiple antibiotics against Staphylococcus aureus was determined. The results showed that the minimum inhibitory concentration of ampicillin was 5 µg / mL, the minimum inhibitory concentration of kanamycin was 9 µg / mL, and the minimum inhibitory concentration of gentamicin was 9 µg / mL. Subsequently, the original strain of Staphylococcus aureus was divided into 3 parts, one blank original strain. A control group, the original strain and the above three antibiotics were configured into the above three antibiotic solutions with a concentration of 1 / 2 of the minimum inhibitory concentration of the original strain. An experimental group, 10 mg of the combination drug was added to the control group.

[0072] The colony concentrations of the above three groups were 1.0×10 8 The original colonies of Staphylococcus aureus with CFU / mL were passaged 10 times, and the single clones passaged 10 times were picked to detect the minimum inhibitory concentration of ampicillin, kanamycin or gentamicin. The experimental results show that when screening for drug-resistant strains, adding a combination of drugs can delay the development of antibiotic resistance in Staphylococcus aureus. Similarly, it can be inferred that adding a combination of drugs can delay the development of antibiotic resistance in Escherichia coli and Candida albicans.

[0073] Table 6 Effect of olive leaf extract antibacterial compound drugs on delaying the antibiotic resistance of Staphylococcus aureus Group MIC (µg / mL)Ampicillin MIC (µg / mL) Kanamycin MIC (µg / mL)Gentamicin Blank Group 5 9 9 Control group (antibiotics) 176 234 256 Experimental group (antibiotics + combination drugs) 6 10 11 The above examples are only for illustrating the technical concept and technical features of the present invention, and cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an antibacterial composition containing olive leaf extract, which is mainly composed of olive leaf extract, separated and purified by amino magnetic microspheres, and then compounded with other ingredients, characterized in that: The steps include: (1) Preparation of amino magnetic microspheres: First, ferric chloride hexahydrate, ferrous sulfate, ammonia water and oleic acid are reacted to prepare oleic acid-coated ferrosoferric oxide particles, and then magnetic polymer microspheres are prepared by a microemulsion method. Then, the magnetic polymer microspheres are first coated with silicon dioxide on the surface of the magnetic microspheres through a hydrolysis and polycondensation reaction of ethyl orthosilicate, and then amino groups are further connected by introducing an amino silanol condensate. After Soxhlet extraction and vacuum drying, magnetic monodisperse porous microspheres are obtained, namely, the amino magnetic microspheres. (2) Preparation of natural product extracts: The natural products are olive leaf, sophora flavescens, phellodendron amurense, cnidium monnieri, liquorice, isatis root, honeysuckle and aloe vera. Different types of natural products are subjected to reflux extraction using ethanol as a solvent, and the extracts are combined after several reflux extractions. The different types of extracts are subjected to rotary evaporation, concentration and drying to obtain olive leaf extract, sophora flavescens extract, phellodendron amurense extract, cnidium monnieri extract, liquorice extract, isatis root extract, honeysuckle extract and aloe vera extract of certain concentrations. (3) Preparation of an antibacterial composition containing olive leaf extract: The olive leaf extract obtained in step (2) is mixed with any one, two or more of Sophora flavescens extract, Phellodendron amurense extract, Cnidium monnieri extract, Licorice extract, Radix Isatidis extract, Flos Lonicerae extract and Aloe extract according to the formula ratio and subjected to ultrasonic treatment to obtain an extract mixed solution; the amino magnetic microspheres obtained in step (1) are placed in the extract mixed solution for shaking magnetic adsorption, and the amino magnetic microspheres are used to adsorb the active ingredients in the extract, and after reaching adsorption equilibrium, the polymer is magnetically separated to obtain magnetic microspheres adsorbed with the active ingredients; the magnetic microspheres adsorbed with the active ingredients are placed in a desorption solvent for shaking desorption, and the adsorbed active ingredients are desorbed from the magnetic microspheres to obtain a desorption liquid, the desorption liquid is concentrated by rotary evaporation and then vacuum dried to obtain an extract, and finally mixed with an auxiliary agent, dissolved and ultrasonicated to obtain an antibacterial composition containing olive leaf extract.

2. The preparation method according to claim 1, characterized in that: The method for preparing the amino-modified magnetic microspheres in step (1) specifically comprises the following steps: ① Preparation of oleic acid-coated ferroferric oxide particles: Dispersing ferric chloride hexahydrate and ferrous sulfate in deionized water, heating under stirring conditions, then adding ammonia water and oleic acid to react, obtaining oleic acid-coated ferroferric oxide particles, using the oleic acid-coated ferroferric oxide particles as a magnetic source, then dispersing them in styrene, pouring them into a pear-shaped funnel, separating and washing them with deionized water, and obtaining a well-dispersed magnetic fluid; ② Preparation of magnetic polymer microspheres by microemulsion method: The magnetic fluid obtained in step ①, the hydrophobic monomer and the hydrophobic initiator are ultrasonically treated under the protection of ice to obtain an ultrasonic solution; at the same time, the emulsifier, the co-emulsifier and the deionized water are stirred in a three-necked flask, and after being fully stirred to form a uniform emulsion, they are added to the ultrasonic solution and prepolymerized at a certain temperature for a period of time, and then the reaction temperature is increased, and a hydrophilic initiator is added to the system and then polymerized for a period of time. After the reaction is completed, the magnetic balls are cleaned with ethanol and water magnetic separation to obtain magnetic polymer microspheres; ③ Coating silica on the surface of magnetic polymer microspheres: Add distilled water and ethanol to the magnetic polymer microspheres obtained in step ②, perform magnetic stirring under water bath conditions, add methacryloyloxyethyl trimethylammonium chloride to react after heating, cool after the reaction, add ammonia water and ethyl orthosilicate to the system to continue the reaction, stir and cool after the reaction, and use ethanol and distilled water to magnetically separate and wash to obtain magnetic polymer microspheres coated with silica; ④ Connecting amino groups on the surface of magnetic polymer microspheres: The magnetic polymer microspheres with surface coated with silica obtained in step ③ are dispersed in an ethanol solution, then mixed with ammonia water and stirred for a period of time, and then the amino silanol condensate is added. After continuous stirring for a period of time, the temperature is raised to react. After the reaction is completed, ethanol and distilled water are used for magnetic separation and cleaning, and then Soxhlet extraction and vacuum drying are performed to obtain magnetic monodisperse porous microspheres.

3. The preparation method according to claim 2, characterized in that: In step ①, the mass ratio of oleic acid: ammonia water: ferrous sulfate: ferric chloride hexahydrate: deionized water is 1: (3-5): (3-5): (1-4): (5-8), and the mass ratio of oleic acid-coated ferrosoferric oxide particles to styrene is (2-5): (2-6).

4. The preparation method according to claim 2, characterized in that: In step ②, the hydrophobic monomers are selected from methacrylic acid and divinylbenzene in a volume ratio of (10-30): (1-4); the hydrophobic initiator is benzoyl peroxide; the mass ratio of the magnetic fluid, the hydrophobic monomer, and the hydrophobic initiator is (10-30): (10-20): (5-10); the emulsifier is sodium dodecyl sulfate, the co-emulsifier is hexadecanol, and the mass ratio of the emulsifier, the co-emulsifier, and deionized water is (0.05-0.15): (0.4-0.6): (20-30); the mass ratio of the emulsion to the ultrasonic solution is (20-30): (3-10); the hydrophilic initiator is potassium persulfate, and the mass ratio of potassium persulfate to benzoyl peroxide is (3-7): (50-100); the prepolymerization reaction has the following reaction conditions: prepolymerization at 50-65°C for 1-4h, and the reaction temperature is then increased to 66-90°C, and the polymerization reaction has a reaction time of 12-36h.

5. The preparation method according to claim 2, characterized in that: In step ③, the mass ratio of the magnetic polymer microspheres, distilled water and ethanol is (0.02-0.1): (1-4): (8-14), the mass ratio of methacryloyloxyethyl trimethyl ammonium chloride to the magnetic polymer microspheres is (1-3): (2-10), and the volume ratio of ammonia water, tetraethyl orthosilicate and methacryloyloxyethyl trimethyl ammonium chloride is (20-40): (10-30): (10-40); in step ④, the surface is coated with silicon dioxide The mass ratio of the magnetic polymer microspheres dispersed with silica on the surface to the ethanol solution is (0.02-0.1):(1-3), the mass ratio of the magnetic polymer microspheres dispersed with silica on the surface to the ammonia solution is (2-10):(10-20), the amino-silanol condensate is aminopropyltrimethoxysilane or (3-aminopropyl)triethoxysilane, and the mass ratio of the magnetic polymer microspheres dispersed with silica on the surface to the amino-silanol condensate is (20-100):(5-10).

6. The preparation method according to claim 2, characterized in that: In step (2), the olive leaf extract is specifically prepared by the following steps: weighing 20-40 g of shade-dried olive leaves, adding 8-14 times the amount of ethanol with a mass concentration of 45-80%, and reflux extraction at 40-50°C for 2 times, each time for 1-2.5 h; combining the extracts, and concentrating them by rotary evaporation at 40-50°C to 0.1 g / mL; the preparation methods of other extracts are similar to the preparation method of the olive leaf extract.

7. The preparation method according to claim 2, characterized in that: In step (3), the mixed extract solution is placed in a centrifuge tube, and amino magnetic microspheres are added at the same time, and the mixture is continuously shaken in a constant temperature oscillator for magnetic adsorption. The mass ratio of the natural product in the mixed extract solution to the amino magnetic microspheres is (7-14): (2-6), and the conditions for magnetic adsorption are: adsorption temperature 20-35°C, adsorption speed 110-150 rpm, and adsorption time 4-8 h; using a 50-75% ethanol solution as a desorption solvent, and continuously shaking in a constant temperature oscillator for desorption, the mass ratio of the desorption solvent to the amino magnetic microspheres is (200-500): (2-6), and the conditions for desorption are: adsorption temperature 20-35°C, adsorption speed 110-150 rpm, and adsorption time 4-8 h; the rotary evaporation of the desorption liquid refers to the rotary evaporation of the desorption liquid until there is no alcohol taste, and then vacuum drying at 50°C to obtain an extract; the auxiliary agent is any one, two or more of nicotinamide, coenzyme Q10, nootkatone, osmanthus hydrosol, rose hydrosol, and antimicrobial peptides; the antimicrobial peptides mainly act on bacterial cell membranes and are not prone to drug resistance. The amino acid sequence is: (RW)n PPPP (KW) n, n= (2-4).

8. The preparation method according to claim 2, characterized in that: The antibacterial composition containing olive leaf extract comprises the following components in mass ratio: Olive leaf: Sophora flavescens: Phellodendron amurense: Cnidium monnieri: Licorice root: Isatis indigotica: Honeysuckle: Aloe vera: Niacinamide: Coenzyme Q10: Nokatone: Osmanthus hydrosol: Rose hydrosol, antimicrobial peptides = (35-60): (0-20): (0-20): (0-20): (0-20): (0-20): (0-20): (0-20): (0-10): (0-10): (0-10): (0-10): (0-10): (0-6); among which, the concentration of natural product extracts is based on the mass of the natural product initially weighed, and its total weight proportion is not less than 85%. Niacinamide, coenzyme Q10, Nokatone, osmanthus hydrosol, rose hydrosol and antimicrobial peptides are auxiliary ingredients, and the sum of their total weight proportions is not higher than 15%.

9. An antibacterial composition containing olive leaf extract prepared by the preparation method according to any one of claims 1 to 8, wherein the composition is in any pharmaceutical dosage form, such as tablets, capsules, suppositories, gels, ointments, etc.

10. Use of the antibacterial composition containing olive leaf extract according to claim 9 in the preparation of drugs for preventing and treating vulvovaginitis.

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