Ploretin-squalene compound as well as preparation method and application thereof

Abstract

The invention provides a phloretin-squalene compound as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention provides a phloretin-squalene compound. The phloretin-squalene compound comprises phloretin, squalene, an oil phase, an emulsifier and a co-emulsifier, the compound is a microemulsion compound. The squalene and the phloretin are creatively combined, the squalene can relieve the cerebral ischemia-reperfusion injury by improving fatty acid metabolism and increasing the cell antioxidant effect, meanwhile, the squalene serves as an oil phase to remarkably increase the entrapment amount of the phloretin, and the improvement effect of the compound on the cerebral ischemia-reperfusion injury is improved; on the other hand, the preparation method provided by the invention is simple and mild in condition, short in reaction time, safe, stable and beneficial to large-scale production, and has a relatively high popularization value.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to a phloretin-squalene complex and a preparation method and application thereof. Background Art

[0002] At present, stroke is a major chronic disease that seriously endangers the health of the Chinese people. According to screening statistics from relevant departments in 2019, the number of people aged 40 and above in my country who are currently suffering from or have suffered from stroke is about 17.04 million. At present, the main treatment for ischemic stroke is to restore blood reperfusion to the ischemic area as soon as possible so that the brain tissue can regain blood oxygen supply. However, when restoring blood supply, the pathological damage of ischemic tissue and the nervous system is often aggravated, causing irreversible cerebral ischemia-reperfusion injury (CIRI).

[0003] Domestic and foreign studies have shown that phloretin belongs to the dihydrochalcone flavonoids, which has multiple biological activities such as antioxidant, anti-cancer, anti-inflammatory, and immunosuppressive. In addition, phloretin is abundant in the peel and root bark of juicy fruits such as apples and pears, and has a wide range of sources. Phloretin can play an important protective role in cerebral ischemia-reperfusion injury through various pathways. However, studies have shown that the solubility of phloretin in water is only about 0.2mM; more importantly, after phloretin is taken orally, it is metabolized in large quantities in the ileum and excreted through feces, so the bioavailability of phloretin is extremely low. This greatly limits the efficacy of its medicine.

[0004] Squalene is a natural substance extracted from the liver of large sharks in the deep sea environment. It is an open-chain triterpenoid compound. The triterpenoid compound composed of 6 isoprene units has the functions of carrying oxygen, lowering cholesterol, anti-oxidation and detoxification. In recent years, it has been found that squalene can antagonize the toxic damage of glutamate to the brain and has a protective effect on acute cerebral ischemia and hypoxia in mice. Studies have also confirmed that squalene can reduce intracellular iron content, improve fatty acid metabolism, and increase cellular antioxidant effects, thereby reducing cerebral ischemia-reperfusion injury.

[0005] In view of the above-mentioned defects, it is urgent to develop a complex containing phloretin and squalene to better solve the problem of further improving reperfusion injury after cerebral ischemia. Summary of the invention

[0006] The purpose of the present invention is to provide a phloretin-squalene complex to solve the technical problems of low solubility and low bioavailability of phloretin in the prior art and to improve reperfusion injury after cerebral ischemia.

[0007] In order to solve the above technical problems, the present invention provides a phloretin-squalene complex, comprising phloretin, squalene, an oil phase, an emulsifier, and an emulsifier aid;

[0008] The complex is a microemulsion complex.

[0009] Further, the oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, glycerol, isopropyl myristate, soybean oil, and triolein;

[0010] Preferably, the oil phase is polyoxyethylene hydrogenated castor oil;

[0011] The emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span;

[0012] Preferably, the emulsifier is egg yolk lecithin;

[0013] The auxiliary emulsifier is a mixture of one or more of polyethylene glycol 12-hydroxy glycol, polyethylene glycol, and sodium alginate;

[0014] Preferably, the co-emulsifier is polyethylene glycol 12 hydroxy diol.

[0015] Furthermore, the particle size of the composite is 80nm-500nm.

[0016] Furthermore, the proportions of the components in the composite by mass percentage are as follows:

[0017] Phloretin: squalene: oil phase: emulsifier: co-emulsifier = (0.1-5): (1.3-7): (1-5): (1-3): (0.1-2).

[0018] The present invention also provides a method for preparing a phloretin-squalene complex, the specific steps of which are as follows:

[0019] Step 1, adding phloretin, squalene, an oil phase, an emulsifier, and an emulsifier co-into a reaction container in the mass percentage of (0.1-5): (1.3-7): (1-5): (1-3): (0.1-2), and then adding tetrahydrofuran, stirring evenly to obtain a first reaction mixture;

[0020] Preferably, the mass percentages of the components are (0.4-2): (2.6-7): (1-5): (1.2-2): (0.3-1.1); more preferably, 0.4: 6.67: 2.7: 1.3: 0.4;

[0021] Step 2, slowly dripping an aqueous solution of an emulsifier with a concentration of 0.1%-0.5% into the first reaction mixture in step 1, and continuously stirring at a rate of 2000 r / min for 10 minutes to obtain a second reaction mixture;

[0022] Step 3, subjecting the second reaction mixture to ultrasonic treatment, and then subjecting the second reaction mixture to rotary evaporation to remove tetrahydrofuran, to obtain a phloretin-squalene complex;

[0023] The power of the ultrasonic treatment is set to 65-260W; preferably, the ultrasonic treatment power is 130W.

[0024] Further, the oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, glycerol, isopropyl myristate, soybean oil, and triolein; preferably polyoxyethylene hydrogenated castor oil;

[0025] The emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span; preferably, egg yolk lecithin;

[0026] The auxiliary emulsifier is a mixture of one or more of polyethylene glycol 12-hydroxy glycol, polyethylene glycol, and sodium alginate; preferably polyethylene glycol 12-hydroxy glycol.

[0027] Furthermore, the ultrasonic treatment time in step 3 is 5-15 min; more preferably 5 min.

[0028] On the other hand, the present invention also provides an application of the above-mentioned phloretin-squalene complex or the complex obtained according to the above-mentioned preparation method, which is used to improve neurological dysfunction, reduce the area of ​​cerebral infarction, reduce the degree of brain tissue edema, and resist oxidative stress damage.

[0029] Furthermore, the administration method in the application is selected from oral administration and intravenous injection.

[0030] By adopting the above technical solution, the present invention has the following beneficial effects:

[0031] The present invention provides a phloretin-squalene complex, which creatively combines squalene with phloretin. Squalene can reduce cerebral ischemia-reperfusion injury by improving fatty acid metabolism and increasing the antioxidant effect of cells. At the same time, squalene also significantly increases the entrapment amount of phloretin as an oil phase, thereby enhancing the improvement effect of the complex on reducing cerebral ischemia-reperfusion injury. On the other hand, the preparation method provided by the present invention has simple and mild conditions, short reaction time, safety, stability, is conducive to large-scale production, and has high promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1It is a schematic diagram of the physical object containing phloretin-squalene complex;

[0034] Figure 2 is the particle size distribution diagram of phloretin-squalene complex;

[0035] Figure 3 is the surface charge distribution diagram of the phloretin-squalene complex;

[0036] Figure 4 The morphology of phloretin-squalene complex under transmission electron microscopy;

[0037] FIG5( a ) is a line graph showing the release process of free phloretin and phloretin-squalene complex in phosphate buffer at pH 7.4;

[0038] FIG5( b ) is a line graph showing the release process of free phloretin and phloretin-squalene complex in artificial gastric juice at pH 1.2;

[0039] FIG5( c ) is a line graph showing the release process of free phloretin and phloretin-squalene complex in artificial intestinal fluid at pH 6.8;

[0040] FIG6( a ) is a line graph showing the changes in particle size and polydispersity index (PDI) of the phloretin-squalene complex at room temperature of 25° C.;

[0041] Figure 6(b) is a line graph showing the changes in particle size and polydispersity index (PDI) of the phloretin-squalene complex at 4°C;

[0042] Figure 7 This is a bar graph of neurological function scores of SD rats after intragastric administration of different drugs;

[0043] Figure 8 This is a schematic diagram of the comparison of cerebral infarction volumes in SD rats after intragastric administration of different drugs;

[0044] Fig. 9 This is a bar graph comparing the cerebral infarction volume of SD rats after intragastric administration of different drugs;

[0045] Fig.10 (a) is a bar graph showing the level of inflammatory factor IL-6 in the serum of SD rats after intragastric administration of different drugs;

[0046] Fig.10 (b) is a bar graph showing the level of inflammatory factor IL-1β in the serum of SD rats after intragastric administration of different drugs;

[0047] Fig.10 (c) is a bar graph showing the level of inflammatory factor I NF-α in the serum of SD rats after intragastric administration of different drugs;

[0048] Fig.11Schematic diagram of biodistribution of phloretin-squalene complex;

[0049] Fig.12 (a) is a bar graph comparing the white blood cell content in the blood of SD rats after intragastric administration of different drugs;

[0050] Fig.12 (b) is a bar graph comparing the red blood cell content in the blood of SD rats after intragastric administration of different drugs;

[0051] Fig.12 (c) is a bar graph comparing the platelet content in the blood of SD rats after intragastric administration of different drugs;

[0052] Fig.12 (d) is a bar graph comparing the monocyte content in the blood of SD rats after intragastric administration of different drugs;

[0053] Fig.12 (e) is a bar graph comparing the lymphocyte content in the blood of SD rats after intragastric administration of different drugs;

[0054] Fig.12 (f) is a bar graph comparing the granulocyte content in the blood of SD rats after intragastric administration of different drugs;

[0055] Fig.13 Schematic diagram of the status of various organs in SD rats after oral administration of different drugs. DETAILED DESCRIPTION

[0056] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0057] The present invention is further explained below in conjunction with specific implementation modes.

[0058] The method for preparing the phloretin-squalene complex provided by the present invention comprises the following specific steps:

[0059] Step 1, adding phloretin, squalene, an oil phase, an emulsifier, and an emulsifier co-into a reaction container in the mass percentage of (0.1-5): (1.3-7): (1-5): (1-3): (0.1-2), and then adding tetrahydrofuran, stirring evenly to obtain a first reaction mixture;

[0060] Step 2, slowly dripping an aqueous solution of an emulsifier with a concentration of 0.1% to 0.5% into the first reaction mixture, and continuously stirring at a rate of 2000 r / min for 10 min to obtain a second reaction mixture;

[0061] Step 3, subjecting the second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 65-260 W and the ultrasonic time set to 5-15 min, and then subjecting the reaction mixture to rotary evaporation to remove tetrahydrofuran to obtain a phloretin-squalene complex.

[0062] Example 1

[0063] like Figure 1-Figure 4 As shown, the phloretin-squalene complex is prepared according to the method provided by the present invention.

[0064] Step 1, weigh 10.33 mg of phloretin, 34.7 mg of egg yolk lecithin, 158.7 mg of squalene, and 75.1 mg of polyoxyethylene hydrogenated castor oil / RH40, respectively, add them into a centrifuge tube, then add 300 uL of tetrahydrofuran as a solvent, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain a first reaction mixture;

[0065] Step 2, slowly dripping 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol into the first reaction mixture, and continuously stirring at a rate of 2000 r / min for 5 min to obtain a second reaction mixture;

[0066] Step 3, subjecting the second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time being 5 min, and then subjecting the reaction mixture to rotary evaporation to remove tetrahydrofuran, to obtain Figure 1 Phloretin-squalene complex shown.

[0067] Zetasizer Nano ZS90 laser particle size analyzer produced by Malvern Instruments, UK, was used to measure the particle size and surface charge of the microemulsion:

[0068] Take 50uL of the phloretin-squalene complex obtained in this example, dilute it to 10mL with ultrapure water, put it into the sample cell for detection, and set the measurement temperature to 25°C. The measurement results are as follows Figure 2-Figure 3 As shown, the particle size of the prepared sample is between 136 nm, the potential is -44 mV, the particle polydispersity coefficient PD I is less than 0.3, and the distribution is uniform.

[0069] Microscopic morphology observation experiment:

[0070] The phloretin-squalene complex prepared in this example was added dropwise to a copper mesh, allowed to stand for 3 min, then stained with 2% phosphotungstic acid for 2 min, and excess dye on the copper mesh was removed with filter paper. The sample was dried at room temperature and observed using a transmission electron microscope at 200 kV. Figure 4 As shown, the particle size is uniform and the shape is regular.

[0071] Determination of phloretin encapsulation efficiency:

[0072] Take the phloretin-squalene complex obtained in this example, separate the unencapsulated phloretin through a glucose gel G50 column, and collect the filtrate; take 100uL of the filtrate, add 0.6mL of anhydrous ethanol, 0.5mL of tetrahydrofuran, and 0.3mL of polyethylene glycol octylphenyl ether to break the emulsion, vortex and centrifuge to take the supernatant. The phloretin content was detected by high performance liquid chromatography, and the encapsulation rate was calculated to be 84.58±3.03%, and the drug loading can reach 4.32±6.2%.

[0073] Example 2

[0074] This example verifies the effect of squalene on the stability of the obtained phloretin-squalene complex.

[0075] Step 1, weighing 10 mg of phloretin, 30 mg of egg yolk lecithin, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively, adding them into a centrifuge tube, repeating the weighing to obtain two identical centrifuge tubes, one centrifuge tube without squalene, and one centrifuge tube with 150 mg of squalene, and then adding 300 uL of tetrahydrofuran as a solvent, vortexing to dissolve and mix, stirring at 2000 r / min for 10 min, to obtain two groups of first reaction mixtures;

[0076] Step 2, taking 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol and slowly dropping it into the two groups of first reaction mixtures in step 1, respectively, and continuously stirring at a rate of 2000 r / min for 5 min to obtain two groups of second reaction mixtures;

[0077] Step 3, subjecting the second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining two groups of complexes containing phloretin.

[0078] The results of the static experiment showed that a group of complexes containing phloretin without the addition of squalene could not form a stable nanoemulsion; while for another group of complexes with the addition of squalene, the phloretin encapsulation amount was significantly improved due to the combined effect of squalene and oil phase compounds, and the nanoemulsion had good stability, which was convenient for industrial storage and practical application.

[0079] Example 3

[0080] This example verifies the effect of different added amounts of squalene on the properties of the obtained phloretin-squalene complex.

[0081] Step 1, weigh 10 mg of phloretin, 60 mg of polyoxyethylene hydrogenated castor oil / RH40, and 30 mg of egg yolk lecithin, add them into a centrifuge tube, repeat the weighing to obtain three portions, add 100 mg, 150 mg, and 200 mg of squalene respectively, and then add 300 uL of tetrahydrofuran as a solvent to each, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0082] Step 2, taking 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol, slowly dripping each of the three groups of first reaction mixtures obtained in step 1, and continuously stirring at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0083] Step 3, subjecting the second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the reaction mixture to rotary evaporation to remove tetrahydrofuran, to obtain three groups of phloretin-squalene complexes.

[0084] 50uL of each of the three groups of phloretin-squalene complexes prepared in this example were taken, diluted to 10mL with ultrapure water, and the above samples were placed in the sample pool for detection, and the measurement temperature was set to 25°C. The effective particle size and PDI data of each group of complexes were measured as shown in Table 1 below:

[0085] Table 1 Effect of different addition amounts of squalene on the effective particle size and PDI of the obtained composites

[0086] Squalene (mg) Effective particles Diameter(nm) PDI 100 91.28±2 .36 0.348±0.01 150 106.00± 1.01 0.250±0.01 200 109.28± 1.07 0.289±0.01

[0087] As can be seen from Table 1, the PD I of the phloretin-squalene complex prepared by adding 100 mg of squalene is greater than 0.3, which does not meet the requirements; when the amount of squalene added is 150 mg and 200 mg, there is no significant difference in the particle size and PD I of the phloretin-squalene complex obtained. From an economic point of view, the ratio of phloretin: squalene: oil phase: emulsifier: co-emulsifier in step 1 of the preparation method provided by the present invention is preferably (0.1-5): 6.67: (1-5): (1-3): (0.1-2).

[0088] Example 4

[0089] This example verifies the effect of different added amounts of phloretin on the stability of the obtained phloretin-squalene complex.

[0090] Step 1, weigh 150 mg of squalene, 60 mg of polyoxyethylene hydrogenated castor oil / RH40, and 30 mg of egg yolk lecithin, add them into a centrifuge tube, repeat the weighing to obtain two portions, add 10 mg and 20 mg of phloretin respectively, and then add 300 uL of tetrahydrofuran as a solvent, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain two groups of first reaction mixtures;

[0091] Step 2, taking 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol, respectively, and slowly dropping them into the two groups of first reaction mixtures obtained in step 1, and continuously stirring at a rate of 2000 r / min for 5 min to obtain two groups of second reaction mixtures;

[0092] Step 3, subjecting the obtained second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the obtained second reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining two groups of phloretin-squalene complexes.

[0093] Take 50uL of each of the two groups of phloretin-squalene complexes prepared in this example, dilute to 10mL with ultrapure water, and place the above samples in the sample pool for detection, and the measurement temperature is set to 25°C. The effective particle size and PDI data of each group of complexes are shown in Table 2 below:

[0094] Table 2 Effects of different amounts of phloretin on the effective particle size and PDI of the obtained composites

[0095] Phloretin(mg) Effective particle size (nm) PDI 10 100.35±1.13 0.256±0.01 20 139.35±0.44 0.126±0.01

[0096] As can be seen from Table 2, the amount of phloretin added has no obvious effect on the effective particle size and PDI of the obtained complex. The effective particle size and PDI of the two groups of complexes obtained in this embodiment can meet the experimental requirements. The results of the static experiment show that in this embodiment, when the amount of phloretin added is 20 mg, the obtained phloretin-squalene complex has drug precipitation after standing for 8 hours at 4°C; while the phloretin-squalene complex obtained in the other group of phloretin added is 10 mg, and the drug precipitation phenomenon never occurs. Therefore, in the preparation method of the phloretin-squalene complex provided by the present invention, the ratio of phloretin: squalene: oil phase: emulsifier: co-emulsifier is preferably 0.4:6.67:(1-5):(1-3):(0.1-2).

[0097] Example 5

[0098] This example verifies the effect of different types of oil phase compounds on the stability of the obtained phloretin-squalene complex.

[0099] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, and 30 mg of egg yolk lecithin, add them into a centrifuge tube, repeat the weighing to obtain three portions, add 101.0 mg of glycerol, 103.4 mg of isopropyl myristate, and 60.7 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively, and then add 300 uL of tetrahydrofuran as a solvent, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0100] Step 2, slowly dripping 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol into the first reaction mixture obtained in step 1, and continuously stirring at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0101] Step 3, subjecting the obtained second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the obtained second reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining three groups of phloretin-squalene complexes.

[0102] 50uL of each of the three groups of phloretin-squalene complexes prepared in this example were taken, diluted to 10mL with ultrapure water, and the above samples were placed in the sample pool for detection, and the measurement temperature was set to 25°C. The effective particle size and PDI data of each group of complexes were measured as shown in Table 3 below:

[0103] Table 3 Effect of adding different types of oil on the effective particle size and PDI of the obtained composites

[0104] oil phase Effective particle size (nm) PDI Glycerol 142.91±1.22 0.249±0.01 Isopropyl myristate 117.81±2.35 0.266±0.01 Polyoxyethylene hydrogenated castor oil 133.22±5.87 0.273±0.05

[0105] As shown in Table 3, the type of oil phase compound has no obvious effect on the effective particle size and PDI of the obtained phloretin-squalene complex. The effective particle size and PDI of the three groups of phloretin-squalene complexes obtained in this embodiment all meet the experimental requirements. The results of the static experiment show that the phloretin-squalene complex prepared with glycerol and isopropyl myristate as the oil phase has precipitation after standing, while the phloretin-squalene complex prepared with polyoxyethylene hydrogenated castor oil as the oil phase has good stability and no precipitation occurs. Therefore, the oil phase material in the preparation method provided by the present invention is preferably polyoxyethylene hydrogenated castor oil / RH40.

[0106] Example 6

[0107] This example verifies the effect of different addition amounts of oil phase compounds on the stability of the obtained phloretin-squalene complex.

[0108] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, and 30 mg of egg yolk lecithin, add them into a centrifuge tube, repeat the weighing to obtain three portions, add 30 mg, 60 mg, and 90 mg of polyoxyethylene hydrogenated castor oil / RH40, respectively, and then add 300 uL of tetrahydrofuran as a solvent, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0109] Step 2, taking 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol, slowly dripping each of the three groups of first reaction mixtures obtained in step 1, and continuously stirring at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0110] Step 3, subjecting the obtained second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the obtained second reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining three groups of phloretin-squalene complexes.

[0111] It can be seen from the observation that in this embodiment, when the amount of polyoxyethylene hydrogenated castor oil added is 30 mg and 90 mg, a small amount of precipitation has occurred when the reaction mixture is subjected to rotary evaporation during the preparation process, while when the amount of polyoxyethylene hydrogenated castor oil added is 60 mg, no precipitation has occurred during the preparation process and subsequent static experiments. Therefore, in the preparation method of the phloretin-squalene complex provided by the present invention, the ratio of phloretin: squalene: oil phase: emulsifier: co-emulsifier is preferably 0.4: 6.67: 2.7: (1-3): (0.1-2).

[0112] Example 7

[0113] This example verifies the effect of different types of emulsifiers on the stability of the obtained phloretin-squalene complex.

[0114] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively and add them into a centrifuge tube, repeat the weighing to obtain two identical centrifuge tubes, add 30.3 mg of soybean lecithin and 30.5 mg of egg yolk lecithin to the two centrifuge tubes respectively, and then add 300 uL of tetrahydrofuran as a solvent, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain two groups of first reaction mixtures;

[0115] Step 2, slowly dripping 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol into the first reaction mixture obtained in step 1, and continuously stirring at a rate of 2000 r / min for 5 min to obtain two groups of second reaction mixtures;

[0116] Step 3, subjecting the obtained second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the obtained second reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining two groups of phloretin-squalene complexes.

[0117] It was observed that the phloretin-squalene complex prepared with soybean lecithin as an emulsifier had a small amount of precipitation after rotary evaporation; while the phloretin-squalene complex prepared with egg yolk lecithin as an emulsifier formed a stable nanoemulsion during the preparation process and the static experiment, and no precipitation occurred. Therefore, in the preparation conditions given in the present invention, the emulsifier is preferably egg yolk lecithin.

[0118] Example 8

[0119] This example verifies the effect of different types of co-emulsifiers on the stability of the obtained phloretin-squalene complex.

[0120] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, 30 mg of egg yolk lecithin, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 and add them into a centrifuge tube, repeat the weighing to obtain three portions, add 300 uL of tetrahydrofuran as a solvent to each portion, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0121] Step 2, under stirring conditions, slowly dripping 2 ml of 0.5% polyethylene glycol 400 aqueous solution, 0.5% sodium alginate, and 0.5% polyethylene glycol 12-hydroxydiol aqueous solution into the three groups of first reaction mixtures, respectively, and after the dropwise addition is completed, stirring is continued at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0122] Step 3, subjecting the second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the reaction mixture to rotary evaporation to remove tetrahydrofuran, to obtain three groups of phloretin-squalene complexes.

[0123] The results of the static experiment show that the two groups of phloretin-squalene complexes prepared with polyethylene glycol 400 and sodium alginate as co-emulsifiers have different degrees of precipitation, while the phloretin-squalene complex prepared with polyethylene glycol 12-hydroxydiol as the co-emulsifier can form a stable microemulsion complex without precipitation. Therefore, in the preparation method provided by the present invention, the co-emulsifier is preferably polyethylene glycol 12-hydroxydiol.

[0124] Example 9

[0125] This example verifies the effect of different concentrations of the co-emulsifier on the stability of the obtained phloretin-squalene complex.

[0126] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, 30 mg of egg yolk lecithin, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 and add them into a centrifuge tube, repeat the weighing to obtain three portions, add 300 uL of tetrahydrofuran as a solvent to each portion, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0127] Step 2, under stirring conditions, slowly dripping 2 ml of aqueous solutions of polyethylene glycol 12-hydroxydiol with concentrations of 0.1%, 0.25%, and 0.5% into the three groups of first reaction mixtures, respectively, and after the dripping is completed, stirring is continued at a rate of 2000 r / min for 5 minutes to obtain three groups of second reaction mixtures;

[0128] Step 3, subjecting the obtained second reaction mixture to ultrasonic treatment, with the ultrasonic power set to 130 W and the ultrasonic time set to 5 min, and then subjecting the obtained second reaction mixture to rotary evaporation to remove tetrahydrofuran, thereby obtaining three groups of phloretin-squalene complexes.

[0129] The three groups of phloretin-squalene complexes obtained in this example were left to stand at 4°C for 10 days. The phloretin-squalene complexes obtained in the experimental groups with the co-emulsifier concentration of 0.1% and 0.25% all had different degrees of precipitation; the phloretin-squalene complexes obtained in the experimental group with the co-emulsifier concentration of 0.5% were not observed to have precipitation. Therefore, in the preparation method provided by the present invention, the concentration of the aqueous solution of the co-emulsifier polyethylene glycol 12-hydroxydiol is preferably 0.5%.

[0130] Example 10

[0131] This example verifies the effect of ultrasonic treatment power on the particle size and PDI of the obtained phloretin-squalene complex.

[0132] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, 30 mg of egg yolk lecithin, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 and add them into a centrifuge tube, repeat the weighing to obtain three portions, add 300 uL of tetrahydrofuran as a solvent to each portion, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0133] Step 2, under stirring conditions, slowly dripping 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol into the three groups of first reaction mixtures respectively, and after the dripping is completed, continuously stirring at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0134] Step 3, subjecting the three groups of second reaction mixtures to ultrasonic treatment, with the ultrasonic powers set to 65 W, 130 W, and 260 W, respectively, and the ultrasonic time being 5 min, and then performing rotary evaporation to remove tetrahydrofuran to obtain three groups of phloretin-squalene complexes.

[0135] The effective particle sizes of the three groups of composites were determined:

[0136] Take 50uL of each of the three groups of phloretin-squalene complexes prepared in this example, dilute to 10mL with ultrapure water, and place the three groups of samples in the sample pool for detection, and the measurement temperature is set to 25°C. The effective particle size and PDI data of the complex obtained in this example are shown in Table 4:

[0137] Table 4 Effective particle size and PDI measurement results of the three groups of composites obtained in Example 10

[0138] Ultrasonic power (W) Effective particle size (nm) PDI 65 163.5±2.9 0.163±0.01 130 137.4±1.3 0.193±0.02 260 165.6±0.6 0.203±0.02

[0139] As can be seen from Table 4, when the ultrasonic power is 130 W, the particle size is small and the PDI is less than 0.3, but when the ultrasonic power is increased or decreased, the particle size increases to varying degrees. Therefore, the power of ultrasonic treatment in the preparation method provided by the present invention is preferably 130 W.

[0140] Embodiment 11

[0141] This example verifies the effect of ultrasonic treatment time on the particle size and PDI of the obtained phloretin-squalene complex.

[0142] Step 1, weigh 10 mg of phloretin, 150 mg of squalene, 30 mg of egg yolk lecithin, and 60 mg of polyoxyethylene hydrogenated castor oil / RH40 and add them into a centrifuge tube, repeat the weighing to obtain three portions, add 300 uL of tetrahydrofuran as a solvent to each portion, vortex to dissolve and mix, and stir at 2000 r / min for 10 min to obtain three groups of first reaction mixtures;

[0143] Step 2, under stirring conditions, slowly dripping 2 ml of a 0.5% aqueous solution of polyethylene glycol 12-hydroxydiol into the three groups of first reaction mixtures respectively, and after the dripping is completed, continuously stirring at a rate of 2000 r / min for 5 min to obtain three groups of second reaction mixtures;

[0144] Step 3, subjecting the three groups of second reaction mixtures to ultrasonic treatment, with the ultrasonic power set to 130 W, the ultrasonic time set to 5 min, 10 min, and 15 min, respectively, and then subjecting the three groups of phloretin-squalene complexes to rotary evaporation to remove tetrahydrofuran.

[0145] Take 50uL of each of the three groups of phloretin-squalene complexes prepared in this example, dilute to 10mL with ultrapure water, and place the three groups of complexes in the sample pool for detection, and the measurement temperature is set to 25°C. The effective particle size and PD I data of the obtained complexes are shown in Table 5:

[0146] Table 5 Effective particle size and PDI measurement results of the three groups of composites obtained in Example 11

[0147] Ultrasonic time (min) Average particle size (nm) PDI 5 127.96±3.0 0.272±0.05 10 133.83±0.9 0.282±0.01 15 129.37±2.9 0.290±0.01

[0148] It can be seen from Table 5 that different ultrasonic times have no significant effect on the particle size and PD I of the obtained phloretin-squalene complex. Considering economic benefits, the present invention recommends that the ultrasonic time in the preparation process is preferably set to 5 min.

[0149] Experimental Example 1

[0150] like Figure 5(a)-Figure 5(c) As shown, this experimental example evaluates the drug release performance of the phloretin-squalene complex provided by the present invention.

[0151] Step A1, taking the phloretin-squalene complex and the reference phloretin solution and placing them in a dialysis bag with a molecular weight of 8000Da, fixing and sealing, and preparing 9 portions in parallel for standby use;

[0152] Step A2: 500 ml of each of the following three solutions were used as dissolution media:

[0153] A. Take phosphate buffer containing 10% ethanol, pH 7.4;

[0154] B. Artificial gastric juice (pH 1.2) prepared in accordance with the 2020 edition of the Chinese Pharmacopoeia;

[0155] C. Artificial intestinal fluid with a pH of 6.8 was used as the dissolution medium. Three parallel tests were performed for each medium.

[0156] Step A2, sampling the phloretin-squalene complex and the reference phloretin solution at 37°C and 120 r / min at 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, and 48 h, respectively, sampling 1 mL at each time point, and immediately adding 1 mL of dissolution medium;

[0157] Step A3, after the collected samples are filtered with a 0.22 μm filter head, the dissolution of the drug is detected by high performance liquid chromatography. The high performance liquid chromatography detection conditions are: chromatographic column: Kromas il 100-5-C18, 4.6×250 nm mobile phase ratio: (acetonitrile: water = 50%: 50%), detection wavelength: 280 nm, flow rate: 1 mL / min.

[0158] From the experimental results, it can be seen that the release of the sample in ethanol-phosphate buffer is shown in Figure 5(a). The free phloretin has a cumulative release of 70% in 16 hours and is almost completely released in 48 hours; while the phloretin-squalene complex has a cumulative release of 50% in 16 hours and enters a plateau phase. The cumulative release is still only about 60% at 48 hours. Figure 5(b)-Figure 5(c) As shown, in the gastric and intestinal fluids, the release rate of free phloretin is also relatively fast, and the cumulative release reaches 100% in 12 hours; in contrast, the release rate of the phloretin-squalene complex provided by the present invention in the gastric and intestinal fluids is significantly slower, but faster than the release rate in ethanol-phosphate buffer, and the cumulative release can reach 80% in 12 hours.

[0159] Experimental Example 2

[0160] like Figure 6(a)-Figure 6(b) As shown, this experimental example verifies the stability of the phloretin-squalene complex.

[0161] Two groups of excess phloretin-squalene complexes provided by the present invention were placed in transparent plastic tubes, sealed and stored at room temperature and 4°C for 120 days, and samples were taken at 1, 2, 3, 5, 7, 10, 15, 20, 30, 60, 90, and 120 days, and the changes in the appearance of the samples were observed and recorded for comparison. The results showed that during the monitoring period, the phloretin-squalene complex did not precipitate or stratify; Figure 6(a)-Figure 6(b) As shown, no significant changes were found in the particle size and PDI of each sample. This experimental example proves that the phloretin-squalene complex has good stability.

[0162] Experimental Example 3

[0163] like Figure 7-Figure 10 As shown in (c), this experimental example verifies the improving effect of phloretin-squalene complex on cerebral ischemia / reperfusion injury.

[0164] Step B1, 40 SPF SD male rats weighing 200-250 g were taken and divided into groups using a simple random method: sham operation group, model group, model + free phloretin group, model + squalene group, model + phloretin-squalene complex group.

[0165] Step B2, establish a rat focal cerebral ischemia / reperfusion injury model by the suture embolism rat cerebral ischemia model method. During the operation, the cerebral blood flow of SD rats was detected by a laser Doppler blood flow monitor to determine whether the sham operation group model was successfully established. Each experimental group was gavaged 3h, 17h, and 26h after modeling, and free phloretin and squalene and phloretin-squalene complex were given respectively. The sham operation group and the model group were given an equal volume of normal saline. After 3 days of feeding, the Longa 5-point scoring standard was used for neurological function scoring, and the scoring standard is shown in Table 4 below:

[0166] Table 5 Scoring criteria for neurological deficits

[0167] score Neurobehavioral changes 0 points No neurological impairment, walking in a nearly straight line 1 point Mild neurological impairment, incomplete extension of the contralateral forepaw, and walking in a tilted manner 2 points Moderate neurological impairment, walking with a leaning posture toward the opposite side (turning in a large circle) 3 points Severe neurological impairment, walking almost centered on the hind limbs (turning in small circles) 4 points Unable to walk spontaneously, or even lose consciousness

[0168] Rating results Figure 7 As shown, the neurological function scores of rats treated with phloretin-squalene complex by oral gavage were significantly lower than those of the model group treated with only normal saline by oral gavage, proving that the neurological function deficits of rats treated with phloretin-squalene complex by oral gavage were significantly alleviated.

[0169] Step B3, take the rats from each experimental group that have undergone the scoring experiment in step B2, and use 2% sodium pentobarbital anesthetic solution for intraperitoneal injection anesthesia, quickly peel off the SD rat brain tissue on ice, fix it in a -20°C refrigerator for 20 minutes, slice it, put it in 2,3,5-triphenyltetrazolium chloride (TTC) staining solution, react at 37°C for 60 minutes, and after confirming that the staining is sufficient, put the brain tissue slices into 4% paraformaldehyde solution, put them in a 4°C refrigerator for fixation for 48 hours, and then take pictures. The results are as follows Figure 8-Figure 9 As shown, it can be clearly seen by comparison that the cerebral infarction volume of rats in the phloretin-squalene complex group was significantly reduced compared with other experimental groups.

[0170] Step B4, take the SD rats that have undergone functional scoring in step B2, collect blood from the orbital vein, let it stand for 3 hours, centrifuge it at a speed of 3000r / min and a temperature of 4°C for 20 minutes, take the upper serum, and determine the inflammatory factors in the serum by enzyme-linked immunosorbent assay. The specific test method is as follows:

[0171] First, take the protein-coated ELISA plate, set up standard wells, sample wells, and blank wells. No sample or ELISA reagent is added to the blank control wells, and 50uL of standard wells of different concentrations are added to each standard well; first add 40uL of sample diluent to the sample to be tested on the ELISA coated plate, and then add 10uL of the sample to be tested, and finally dilute it to 5 times the sample. Add the sample to the bottom of the ELISA plate well, try not to touch the well wall, and gently shake to mix. Add 100uL of ELISA reagent to each well, except for the blank well. Seal the plate with a sealing film and incubate at 37℃ for 60min. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30s and discard it, repeat this 5 times, and pat dry. Then add the color developer to each well, gently shake and mix, and color at 37℃ in the dark for 15 minutes. Add 50μL of stop solution to each well to stop the reaction, adjust the blank well to zero, and measure the absorbance of each well in sequence at a wavelength of 450nm, that is, the OD value.

[0172] Test results such as Fig.10 (a)- Fig.10 As shown in (c), after oral administration of phloretin-squalene complex, the inflammatory factors IL-6, IL-1β, and INF-α in rats were significantly reduced, which is more conducive to alleviating cerebral ischemia-reperfusion injury.

[0173] Experimental Example 4

[0174] like Fig.11 As shown, this experimental example verifies the distribution of the phloretin-squalene complex in the body.

[0175] Step C1, SD rats were randomly divided into groups, and were gavaged with nanoemulsion loaded with Di D, phloretin-squalene complex, and free phloretin, respectively. The rats were killed 3h and 6h later, and the hearts, livers, and brains were removed and washed with normal saline.

[0176] Step C2, dry the tissue with filter paper and image it with Berthold animal in vivo imaging system. Fig.11 As shown, compared with free phloretin, the fluorescence signal of the phloretin-squalene complex in brain tissue is enhanced. This experimental example proves that the phloretin-squalene complex is more likely to accumulate in brain tissue, which is beneficial to improve reperfusion injury after cerebral ischemia.

[0177] Experimental Example 5

[0178] like Fig.12 (a)- Fig.13 As shown, this experimental example verifies the safety of the phloretin-squalene complex.

[0179] Step D1, after one week of adaptive feeding, SD rats were randomly divided into 3 groups (n=3) and intragastrically treated with physiological saline, phloretin solution and phloretin-squalene complex at a dose of 5 mg / kg for 3 consecutive days.

[0180] Step D2: On the 5th day, the mice were killed, and blood was collected from the abdominal aorta using a fully automatic blood cell analyzer for veterinary testing. The main tests included platelets, red blood cells, white blood cells, lymphocytes, neutrophils, and monocytes. The test results are as follows: Fig.12 (a)- Fig.12 As shown in (f), the blood cell test results of rats after oral administration of phloretin-squalene complex were all within the normal range, indicating safety in blood items.

[0181] Step D3, the heart, liver, spleen, lung, kidney, and intestine of each group of experimental rats were subjected to H&E staining and analyzed under an inverted fluorescence microscope. Fig.13 As shown, the results of H&E staining showed that there was no obvious lesion in various organs of rats after oral administration of phloretin-squalene complex, indicating that it has good biological safety.

[0182] In summary, the phloretin-squalene complex provided by the present invention has significant effects on improving neurological dysfunction, reducing cerebral infarction area, reducing brain tissue edema, resisting oxidative stress damage, and reducing reperfusion injury after cerebral ischemia; and has good biological safety. On the other hand, the preparation method of the phloretin-squalene complex provided by the present invention is simple, mild, safe and stable, and the obtained product has good stability, which is conducive to large-scale production.

[0183] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A phloretin-squalene complex, characterized in that: The complex comprises phloretin, squalene, an oil phase, an emulsifier, and an emulsifier assistant; The complex is a microemulsion complex.

2. The composite according to claim 1, characterized in that The oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, glycerol, isopropyl myristate, soybean oil, and triolein; The emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span; The auxiliary emulsifier is a mixture of one or more of polyethylene glycol 12-hydroxy glycol, polyethylene glycol and sodium alginate.

3. The composite according to claim 1, characterized in that The particle size of the composite is 80nm-500nm.

4. The composite according to claim 1, characterized in that The mass percentage of each component in the composite is: Phloretin: squalene: oil phase: emulsifier: co-emulsifier = (0.1-5): (1.3-7): (1-5): (1-3): (0.1-2).

5. A method for preparing the composite according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: Step 1, adding phloretin, squalene, an oil phase, an emulsifier, and an emulsifier co-into a reaction container in the mass percentage of (0.1-5): (1.3-7): (1-5): (1-3): (0.1-2), and then adding tetrahydrofuran, stirring evenly to obtain a first reaction mixture; Step 2, slowly dripping an aqueous solution of an emulsifier with a concentration of 0.1% to 0.5% into the first reaction mixture, and continuously stirring at a rate of 2000 r / min for 10 min to obtain a second reaction mixture; Step 3, subjecting the second reaction mixture to ultrasonic treatment, and then subjecting the second reaction mixture to rotary evaporation to remove tetrahydrofuran, to obtain a phloretin-squalene complex; The power of the ultrasonic treatment was set at 65-260W.

6. The preparation method according to claim 5, characterized in that: The mass ratio of phloretin, squalene, oil phase, emulsifier and co-emulsifier in step 1 is (0.4-2):(2.6-7):(1-5):(1.2-2):(0.3-1.1).

7. The preparation method according to claim 5, characterized in that: The oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, glycerol, isopropyl myristate, soybean oil, and triolein; The emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span; The auxiliary emulsifier is a mixture of one or more of polyethylene glycol 12-hydroxy glycol, polyethylene glycol and sodium alginate.

8. The preparation method according to claim 5, characterized in that: The power of the ultrasonic treatment in step 3 was set to 130 W; The ultrasonic treatment time is 5-15 min.

9. Use of the composite according to any one of claims 1 to 4 or the composite obtained by the preparation method according to any one of claims 5 to 8, characterized in that: The application is used to improve neurological dysfunction, reduce the area of ​​cerebral infarction, alleviate the degree of brain tissue edema, and resist oxidative stress damage.

10. The use according to claim 9, characterized in that: The administration method of the application is selected from oral administration and intravenous injection.

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