Composition containing trilobatin and preparation method and application thereof

Abstract

The invention provides a trilobatin-containing composition as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The trilobatin-containing composition provided by the invention comprises trilobatin, squalene, an oil phase and an emulsifier, the composition comprises the following components in percentage by mass: 0.1-5% of trilobatin, 1.3-7% of squalene, 4-10% of an oil phase and 1-3% of an emulsifier, the composition is a microemulsion. The trilobatin and squalene are mixed according to a proper ratio, so that the distribution of the trilobatin in brain tissues can be improved, the solubility can be enhanced, the stability can be improved, the volume of cerebral infarction caused by cerebral ischemia or reperfusion injury can be reduced, the neurological function score can be improved, the level of inflammatory factors in a body can be reduced, and the trilobatin and squalene composition has good safety and stability. On the other hand, the preparation method of the composition provided by the invention has the characteristics of simple process and mild conditions, is beneficial to large-scale production, and has wide application space and market prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composition containing trilobatin, and a preparation method and application thereof. Background Art

[0002] At present, stroke has become the leading cause of death among Chinese residents. With the aging of the population and the acceleration of urbanization, the prevalence of stroke risk factors is obvious. At present, the main response to ischemic stroke is to reperfuse blood to the ischemic area so that the brain tissue can regain blood oxygen supply. However, cerebral ischemia causes brain cell damage, and after blood flow reperfusion is restored, cerebral ischemia-reperfusion injury is likely to occur.

[0003] Trilobatin is one of the main components of the traditional Chinese medicine Polyspicate, and belongs to the dihydrochalcone class of compounds. Studies have shown that trilobatin can significantly inhibit the oxidative stress response during cerebral ischemia, improve the antioxidant level, and thus improve brain tissue damage and brain function scores caused by cerebral ischemia-reperfusion injury. However, trilobatin has poor solubility in water and is rapidly metabolized in the body, so the oral bioavailability of trilobatin is extremely low. This greatly limits the full effect of its active ingredients.

[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. At the same time, studies have confirmed that squalene can reduce intracellular iron content, improve fatty acid metabolism, increase cellular antioxidant effect, and thus reduce cerebral ischemia-reperfusion injury.

[0005] Therefore, it is very necessary to develop a composition containing trilobatin and squalene for improving cerebral ischemia-reperfusion injury and improving the stability and bioavailability of the drug. Summary of the invention

[0006] The purpose of the present invention is to provide a composition containing trilobatin and a preparation method and application thereof, so as to solve the technical problems of poor stability and low bioavailability existing in the prior art.

[0007] In order to solve the above technical problems, the present invention provides a composition containing trilobatin, comprising trilobatin, squalene, an oil phase, and an emulsifier;

[0008] The mass fractions of the components in the composition are 0.1%-5% of trilobatin, 1.3%-7% of squalene, 4%-10% of oil phase and 1%-3% of emulsifier.

[0009] The mass fraction of trilobatin is preferably 0.4%-2%, and more preferably 0.4%;

[0010] The mass fraction of squalene is preferably 2.6%-4.5%, and more preferably 6.7%;

[0011] The mass fraction of the oil phase is preferably 5%-6.5%, and more preferably 2.7%;

[0012] The mass fraction of the emulsifier is preferably 1.2%-2%, and more preferably 1.3%.

[0013] Trilobatin can significantly inhibit the oxidative stress response during cerebral ischemia-reperfusion and improve the antioxidant level, thereby improving brain tissue damage and brain function scores caused by cerebral ischemia-reperfusion injury; squalene can antagonize glutamate, reduce intracellular iron content, improve fatty acid metabolism, increase cellular antioxidant effects, and reduce cerebral ischemia-reperfusion injury.

[0014] The composition is a microemulsion;

[0015] Microemulsions can improve the solubility and physical stability of poorly soluble drugs; they can greatly increase the stability and bioavailability of drugs and reduce toxic side effects.

[0016] Furthermore, the composition also includes an emulsifier;

[0017] The mass fraction of the auxiliary emulsifier in the composition is 0.1-2%, preferably 0.3-1.1%, and more preferably 0.9%.

[0018] Appropriate co-emulsifiers help to form submicron monomer droplets, adsorb most of the emulsifier molecules, and thus enhance the stability of the emulsion.

[0019] Further, the oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, palm oil, olive oil, soybean oil, triolein, glycerol, and isopropyl myristate;

[0020] Preferably, the oil phase is polyoxyethylene hydrogenated castor oil RH40.

[0021] The oil phase can increase the emulsion loading capacity of trilobatin and significantly improve the stability of the microemulsion.

[0022] Furthermore, the emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span;

[0023] Preferably, the emulsifier is egg yolk lecithin.

[0024] Furthermore, the auxiliary emulsifier is a mixture of one or more of Tween-80, polyethylene glycol, and sodium alginate;

[0025] Preferably, the co-emulsifier is Tween-80.

[0026] On the other hand, the present invention also provides a method for preparing the above composition, the specific steps are as follows:

[0027] Step 1, add 0.1-5 parts of trilobatin, 1.3-7 parts of squalene, 4-10 parts of oil phase, and 1-3 parts of emulsifier into a reaction container, then add an organic solvent, and stir at 2000 r / min for 10 minutes to obtain a mixture solution;

[0028] Step 2, taking 0.1-2 parts of a co-emulsifier and adding it into water to obtain a co-emulsifier solution; under continuous stirring, adding the co-emulsifier solution dropwise into the mixture solution, stirring at 2000 r / min for 10 min to obtain colostrum;

[0029] Step 3, subjecting the colostrum to ultrasonic treatment, and removing the organic solvent by rotary evaporation to obtain a trilobatin-squalene microemulsion composition.

[0030] The above components are all calculated by mass.

[0031] Furthermore, the oil phase in step 1 is a mixture of one or more of polyoxyethylene hydrogenated castor oil, palm oil, olive oil, soybean oil, triolein, glycerol, and isopropyl myristate; the emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween, and Span;

[0032] The auxiliary emulsifier in step 2 is a mixture of one or more of Tween-80, polyethylene glycol and sodium alginate.

[0033] The organic solvent in step 1 and step 3 is a mixture of one or more of ethanol, tetrahydrofuran and dichloromethane, preferably tetrahydrofuran.

[0034] Further, the power of the ultrasonic treatment is set to 32.5-487.5W; preferably, the power of the ultrasonic treatment is 65-260W, and more preferably 260W;

[0035] The ultrasonic treatment time is 5-20 min, preferably 5-15 min, and more preferably 5 min.

[0036] The present invention also provides a method for improving brain damage during cerebral ischemia-reperfusion, wherein the method uses a composition containing trilobatin or a trilobatin-squalene microemulsion composition prepared according to the above method.

[0037] Furthermore, the dosage form of the composition is selected from oral dosage form and injection dosage form.

[0038] By adopting the above technical solution, the present invention has the following beneficial effects:

[0039] The composition containing trilobatin provided by the present invention can improve the distribution of trilobatin in brain tissue, reduce the volume of cerebral infarction caused by cerebral ischemia or reperfusion injury, improve neurological function score, reduce the level of inflammatory factors in the body, and has good safety and stability. On the other hand, the preparation method of the above-mentioned composition provided by the present invention has the characteristics of simple process and mild conditions, is conducive to large-scale production, and has broad application space and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] 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.

[0041] Figure 1 A transmission electron microscope scanning image of the trilobatin-squalene microemulsion composition provided in Example 1 of the present invention;

[0042] Figure 2 This is a schematic diagram of the trilobatin-squalene microemulsion composition;

[0043] Figure 3 This is a particle size distribution diagram of the trilobatin-squalene microemulsion composition provided in Example 1;

[0044] Figure 4 The surface charge distribution diagram of the trilobatin-squalene microemulsion composition provided in Example 1;

[0045] Figure 5 A comparison diagram of static precipitation experiments of two groups of trilobatin-squalene microemulsion compositions with different oil phases added provided in Example 4;

[0046] FIG6(a) is a line graph comparing the release amounts of the trilobatin solution control group and the trilobatin-squalene microemulsion composition experimental group in 10% ethanol-phosphate buffer in Example 9;

[0047] FIG6( b ) is a line graph comparing the release amounts of the trilobatin solution control group and the trilobatin-squalene microemulsion composition experimental group in artificial gastric juice in Example 9;

[0048] FIG6(c) is a line graph comparing the release amounts of the trilobatin solution control group and the trilobatin-squalene microemulsion composition experimental group in artificial intestinal fluid in Example 9;

[0049] Figure 7 It is a line graph for monitoring the particle size and PDI changes of the trilobatin-squalene microemulsion composition at room temperature in Example 10;

[0050] Figure 8 It is a line graph for monitoring the particle size and PDI changes of the trilobatin-squalene microemulsion composition at 4° C. in Example 10;

[0051] Fig. 9 This is a comparison chart of the neurological function scores of SD rats after being treated with five groups of different drugs provided in Example 11;

[0052] Fig.10 This is a schematic diagram showing the comparison of cerebral infarction volumes of SD rats after being treated with five groups of different drugs provided in Example 11;

[0053] Fig.11 This is a comparison chart of brain tissue infarction rates of SD rats after being treated with different drugs in Example 11;

[0054] Fig.12 This is a schematic diagram of the level of inflammatory factor tumor necrosis factor-α in SD rats after being treated with the trilobatin-squalene microemulsion composition in Example 11;

[0055] Fig.13 This is a schematic diagram of the level of interleukin-1β, a inflammatory factor, in SD rats after being treated with the trilobatin-squalene microemulsion composition in Example 11;

[0056] Fig.14 This is a schematic diagram of the level of interleukin-6, an inflammatory factor, in SD rats after being treated with the trilobatin-squalene microemulsion composition in Example 11;

[0057] Fig.15 A schematic diagram of the biodistribution of the trilobatin-squalene microemulsion composition provided in Example 12;

[0058] FIG. 16( a ) is a bar graph comparing the content of aspartate aminotransferase, a liver function marker, in SD rats that were gavaged with the trilobatin-squalene microemulsion composition for three consecutive days in Example 13 and in the normal control group;

[0059] FIG16( b ) is a bar graph comparing the liver function marker alanine aminotransferase content in SD rats after continuous gavage of the trilobatin-squalene microemulsion composition for three days in Example 13 and that in the normal control group;

[0060] FIG16( c ) is a bar graph comparing the blood urea nitrogen content, a renal function marker, of SD rats after continuous gavage of the trilobatin-squalene microemulsion composition for three days in Example 13 and the normal control group;

[0061] FIG16( d ) is a bar graph comparing the creatinine content, a renal function marker, in SD rats after continuous gavage of the trilobatin-squalene microemulsion composition for three days in Example 13 and in the normal control group;

[0062] FIG. 17( a ) is a bar graph showing the comparison of the number of white blood cells in SD rats in Example 14 after three days of oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients;

[0063] FIG17( b ) is a bar chart comparing the number of red blood cells in SD rats administered with free trilobatin, trilobatin-squalene microemulsion composition, and excipients three days after oral administration in Example 14;

[0064] FIG. 17( c ) is a comparative bar chart of the platelet counts of SD rats in Example 14 after three days of oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients;

[0065] FIG. 17( d ) is a bar graph showing the percentage of monocytes in SD rats in Example 14 after three days of oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients;

[0066] Figure 17(e) is a bar graph showing the percentage of neutrophils in SD rats in Example 14 after three days of oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients;

[0067] FIG17( f ) is a bar graph showing the percentage of lymphocytes in SD rats after oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients for three days;

[0068] Fig.18 This is a comparative schematic diagram of the H&E staining results of various organs of SD rats in Example 14 three days after oral administration of free trilobatin, trilobatin-squalene microemulsion composition, and excipients. DETAILED DESCRIPTION

[0069] 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.

[0070] The present invention is further explained below in conjunction with specific implementation modes.

[0071] The composition containing trilobatin provided by the present invention comprises, by mass fraction, 0.1-5% of trilobatin, 1.3-7.0% of squalene, 4-10% of oil phase, 1-3% of emulsifier, 0.1-2% of co-emulsifier, and the balance is water. The specific preparation method is as follows:

[0072] Step 1, add 0.1-5 parts of trilobatin, 1.3-7 parts of squalene, 4-10 parts of oil phase, and 1-3 parts of emulsifier into a centrifuge tube, then add tetrahydrofuran, vortex to dissolve and mix, and stir at a rate of 2000 r / min for 5-12 minutes to obtain a mixture solution;

[0073] Step 2, taking 0.1-2 parts of a co-emulsifier and adding it into water to obtain a co-emulsifier solution; under continuous stirring, dropwise adding the co-emulsifier solution into the first mixture solution, stirring at a rate of 2000 r / min for 5-12 min, to obtain colostrum;

[0074] Step 3, subjecting the colostrum to ultrasonic treatment using an ultrasonic cell disruptor, setting the power to 32.5-487.5 W, the ultrasonic time to 5-20 min, and removing tetrahydrofuran by rotary evaporation to obtain a trilobatin-squalene microemulsion composition.

[0075] Example 1

[0076] like Figure 1-Figure 4 As shown, this example verifies that the trilobatin-squalene microemulsion composition is prepared according to the method provided by the present invention.

[0077] Step 1, weigh 10.01 mg of trilobatin, 30.2 mg of egg yolk lecithin, 153.4 mg of squalene, and 62.8 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively and place them in a centrifuge tube, then add 300 uL of tetrahydrofuran, vortex to dissolve and mix, and stir at a rate of 2000 r / min for 10 min to obtain a mixture solution;

[0078] Step 2, prepare a 1% Tween-80 aqueous solution; slowly drop 2 mL of the 1% Tween-80 aqueous solution into the mixture solution obtained in step 1 under continuous stirring;

[0079] After the addition was completed, the mixture was stirred at a rate of 2000 r / min for 5 min to form colostrum;

[0080] Step 3, subjecting the colostrum obtained in step 2 to ultrasonic treatment using an ultrasonic cell disruptor, setting the power to 260 W and the ultrasonic time to 5 min, and then removing tetrahydrofuran by rotary evaporation to obtain a trilobatin-squalene microemulsion composition.

[0081] Microscopic morphology detection of microemulsion composition:

[0082] The trilobatin-squalene microemulsion composition prepared in this example was added dropwise onto a copper mesh and allowed to stand for 3 minutes. The composition was then stained with 2% phosphotungstic acid for 2 minutes, and excess dye was removed from the copper mesh with filter paper. After the sample was dried at room temperature, it was observed under a transmission electron microscope at 200 kV. The microscopic morphology of the sample was as follows: Figure 1 As shown, the physical form is Figure 2 shown.

[0083] Determination of particle size and surface charge of microemulsion composition:

[0084] like Figure 3-Figure 4 As shown, the particle size and surface charge of the microemulsion were measured using a Zetasizer Nano ZS90 laser particle size analyzer (Malvern Instruments, UK):

[0085] Take 50uL of the trilobatin-squalene microemulsion composition prepared in this example, dilute it to 10mL with ultrapure water, and put the above samples into the sample pool for detection, and the measurement temperature is set to 25°C. The test results are as follows: Figure 3-Figure 4 As shown, the particle size of the sample prepared in this example is between 120 nm, the potential is -32.7 mV, and its particle polydispersity coefficient PDI is less than 0.3, indicating that the particle distribution is uniform.

[0086] Determination of trilobatin encapsulation efficiency in trilobatin-squalene microemulsion composition:

[0087] Step S1, taking the trilobatin-squalene microemulsion composition prepared in this example, separating the unencapsulated trilobatin through a glucose gel G50 column, and collecting the filtrate;

[0088] Step S2, take 100uL of the filtrate, add 900uL of methanol, perform ultrasonic demulsification for 30min, let stand for 10min, and then take the supernatant. Use high performance liquid chromatography to detect the content of trilobatin, and calculate that the encapsulation efficiency is 82.8±2.4%, and the drug loading can reach 3.34±0.07%.

[0089] Example 2

[0090] This example verifies the effect of different organic solvents on the dissolution of trilobatin in a microemulsion composition.

[0091] Step 1, weigh three portions of 10 mg of trilobatin into centrifuge tubes, add 0.3 ml of tetrahydrofuran, dichloromethane, and ethanol organic solvents, vortex for 3 minutes to dissolve, and observe the dissolution phenomenon.

[0092] The dissolution of trilobatin by different organic solvents is compared as shown in Table 1 below. No drug precipitation was found in the tetrahydrofuran group.

[0093] Table 1 Example 2 Dissolution of trilobatin by different organic solvents

[0094]

[0095]

[0096] Example 3

[0097] This example verifies the effect of different organic solvents on the dissolution of squalene and egg yolk lecithin in the microemulsion composition.

[0098] Step 1, weigh 30 mg of egg yolk lecithin and 150 mg of squalene in three portions into centrifuge tubes, add 0.3 ml of tetrahydrofuran, ethanol, and dichloromethane respectively, and sonicate for 10 minutes to observe the dissolution phenomenon.

[0099] The dissolution of egg yolk phosphatidylcholine and squalene by different organic solvents is compared as shown in Table 2. No drug precipitation was found in the tetrahydrofuran group. Therefore, the organic solvent in the preparation method provided in the present invention is preferably tetrahydrofuran.

[0100] Table 2 Example 3 Dissolution of egg yolk lecithin and squalene by different organic solvents

[0101] Organic solvents Phenomenon Ethanol Slightly soluble Tetrahydrofuran Dissolve Dichloromethane Slightly soluble

[0102] Example 4

[0103] like Figure 5 As shown, this example verifies the effect of using different emulsifiers on the stability of the microemulsion composition.

[0104] Step 1, weigh 10 mg of trilobatin, 160 mg of squalene, and 62.8 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively and place them in centrifuge tubes, and weigh two centrifuge tubes in total;

[0105] Add 30.3 mg of soybean lecithin and 30.5 mg of egg yolk lecithin to two centrifuge tubes respectively, then add 300 uL of tetrahydrofuran to each tube, vortex to dissolve and mix, and stir at a rate of 2000 r / min for 10 min to obtain a mixture solution;

[0106] Step 2, prepare a 1% Tween-80 aqueous solution; under continuous stirring, slowly drip 2 mL of the 1% Tween-80 aqueous solution into the two tubes obtained in step 1;

[0107] After the addition was completed, the mixture was stirred at a rate of 2000 r / min for 5 min each to form colostrum;

[0108] Step 3, the colostrum obtained in step 2 is subjected to ultrasonic treatment using an ultrasonic cell crusher, with the power set to 260 W and the ultrasonic time set to 5 min, followed by rotary evaporation to remove tetrahydrofuran, to obtain two groups of trilobatin-squalene microemulsion compositions.

[0109] By comparing the two groups of trilobatin-squalene microemulsion compositions obtained in this example, it can be found that Figure 5As shown, the trilobatin-squalene microemulsion composition prepared by adding soybean lecithin in step 1 has drug precipitation after standing for 5 minutes; while the trilobatin-squalene microemulsion composition added with egg yolk lecithin in step 1 has better stability and no drug precipitation is found. Therefore, the emulsifier in the preparation method provided in the present invention is preferably egg yolk lecithin.

[0110] Example 5

[0111] This example verifies the effect of adding different oil phase substances on the stability of the prepared microemulsion composition.

[0112] Step 1, weigh 10 mg of trilobatin, 30 mg of egg yolk lecithin, and 150 mg of squalene respectively, and place them in centrifuge tubes, and weigh three sets of centrifuge tubes in total;

[0113] Add 101 mg of glycerol, 103.5 mg of isopropyl myristate, and 60.8 mg of polyoxyethylene hydrogenated castor oil / RH40 to the three groups of centrifuge tubes respectively; then add 300 uL of tetrahydrofuran to each group, vortex to dissolve and mix, and stir at a rate of 2000 r / min for 10 min to obtain three groups of mixed solutions;

[0114] Step 2, prepare a 1% Tween-80 aqueous solution; slowly drip 2 mL of the 1% Tween-80 aqueous solution into each of the three mixed solutions obtained in step 1 under continuous stirring;

[0115] After the addition was completed, the mixture was stirred at a rate of 2000 r / min for 5 min to form three groups of colostrum;

[0116] Step 3, the colostrum obtained in step 2 is subjected to ultrasonic treatment using an ultrasonic cell crusher, with the power set to 260 W and the ultrasonic time set to 5 min, followed by rotary evaporation to remove tetrahydrofuran to obtain three groups of trilobatin-squalene microemulsion compositions.

[0117] Zetasizer Nano ZS90 laser particle size analyzer (Malvern Instruments, UK) was used to measure the particle size and surface charge of the microemulsion:

[0118] 50uL of each of the three groups of trilobatin-squalene microemulsion compositions prepared in this example were taken, diluted to 10mL with ultrapure water, and placed in the sample pool for detection, with the measurement temperature set at 25°C. The particle sizes and potentials of the three groups of prepared samples are shown in Table 3.

[0119] Table 3 Determination of effective particle size and PDI of Example 5 ( n=3)

[0120] Oil phase substances 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

[0121] It can be seen from Table 3 that the particle polydispersity index (PDI) of the three groups of prepared samples is less than 0.3.

[0122] On the other hand, after the three groups of trilobatin-squalene microemulsion compositions were left standing at room temperature for 6 hours, it was observed that the microemulsion composition with glycerol and isopropyl myristate as the oil phase showed drug precipitation; while the microemulsion composition with polyoxyethylene hydrogenated castor oil / RH40 as the oil phase was stable and no drug precipitation occurred. Therefore, in the preparation method of the trilobatin-squalene microemulsion composition provided by the present invention, the oil phase additive is preferably polyoxyethylene hydrogenated castor oil / RH40.

[0123] Example 6

[0124] This example verifies the effect of adding different co-emulsifiers on the stability of the prepared microemulsion composition.

[0125] Step 1, weigh 10.01 mg of trilobatin, 30.2 mg of egg yolk lecithin, 153.4 mg of squalene, and 62.8 mg of polyoxyethylene hydrogenated castor oil / RH40 respectively and place them in a centrifuge tube, then add 300 uL of tetrahydrofuran, vortex to dissolve and mix, and stir at a rate of 2000 r / min for 10 min to obtain a first mixed solution; repeat step 1 to obtain five groups of mixed solutions;

[0126] Step 2, under continuous stirring, slowly dripping 2 mL of different emulsifiers into the five mixed solutions: 0.5% polyethylene glycol 400 aqueous solution, 0.5% sodium alginate, 0.25% Tween-80 aqueous solution, 0.5% Tween-80 aqueous solution, and 1% Tween-80 aqueous solution;

[0127] After the addition was completed, the mixture was stirred at a rate of 2000 r / min for 5 min to form five groups of colostrum;

[0128] Step 3, subjecting the five groups of colostrum obtained in step 2 to ultrasonic treatment using an ultrasonic cell crusher, setting the power to 260 W and the ultrasonic time to 5 min, and then removing tetrahydrofuran by rotary evaporation to obtain five groups of trilobatin-squalene microemulsion compositions.

[0129] Through visual observation, the group adding 0.5% sodium alginate had precipitation in the process of preparing colostrum; the experimental group adding 0.5% polyethylene glycol 400, the experimental group adding 0.25% Tween-80 aqueous solution and the experimental group adding 0.5% Tween-80 aqueous solution had precipitation after rotary evaporation; and the experimental group adding 1% Tween-80 had no precipitation, and a stable microemulsion composition could be formed. Therefore, the co-emulsifier in the preparation method provided by the present invention is preferably Tween-80, and the concentration is 1%.

[0130] Example 7

[0131] This example verifies the effect of different powers on the microemulsion composition during the ultrasonic treatment of colostrum.

[0132] The preparation conditions of this embodiment are basically the same as those of Example 1, except that, in step 2, three groups of the same colostrum are prepared, and in step 3, the ultrasonic cell pulverizer is used for ultrasonic treatment, and the power is set to 32.5 W, 65 W, 130 W, 260 W, 325 W, and 487.5 W respectively, and the ultrasonic time is 5 min. Then, tetrahydrofuran is removed by rotary evaporation to obtain six groups of trilobatin-squalene microemulsion compositions.

[0133] Take 50uL of each of the six groups of trilobatin-squalene microemulsion compositions prepared in this example, dilute to 10mL with ultrapure water, and put the above samples into sample cells for detection. The particle sizes and PDI of the six groups of samples at 25°C are shown in Table 4:

[0134] Table 4 The particle size and PDI measurement results of the six groups of microemulsion compositions obtained in this example ( n=3)

[0135] Ultrasonic power (W) Effective particle size (nm) PDI 32.5 162.1±2.0 0.201±0.02 65 153.5±2.9 0.198±0.01 130 167.4±1.3 0.213±0.02 260 127.4±1.3 0.183±0.02 325 153.2±1.5 0.209±0.03 487.5 183.44±1.8 0.244±0.03

[0136] The test results in Table 4 show that when the ultrasonic power is 260 W, the particle size is small and the PD I is also low, but when the ultrasonic power is increased or decreased, the particle size will increase. Therefore, the ultrasonic power in the preparation method provided by the present invention is preferably 260 W.

[0137] Example 8

[0138] This example verifies the effect of different treatment times during ultrasonic treatment of colostrum on the microemulsion composition.

[0139] The preparation method of this embodiment is basically the same as that of Example 1, except that four groups of the same colostrum are prepared in step 2, and in the ultrasonic treatment process of step 3, the treatment time is set to 5 min, 10 min, 15 min, and 20 min, respectively, and then the tetrahydrofuran is removed by rotary evaporation to obtain four groups of trilobatin-squalene microemulsion compositions.

[0140] Take 50uL of each of the four groups of trilobatin-squalene microemulsion compositions prepared in this example, dilute to 10mL with ultrapure water, and put the above samples into sample cells for detection. The particle sizes and PDI of the four groups of samples at 25°C are shown in Table 5:

[0141] Table 5 Particle size and PDI measurement results of four groups of microemulsion compositions obtained in this example ( n=3)

[0142] Ultrasound duration (min) Average particle size (nm) PDI 5 121.96±3.0 0.194±0.05 10 132.83±0.9 0.282±0.01 15 159.37±2.9 0.275±0.01 20 212.47±2.0 0.257±0.01

[0143] It can be seen from the test data in Table 5 that the ultrasonic treatment time of 5-20 min has no significant effect on the particle size and PDI of the microemulsion composition. Therefore, from an economic point of view, the ultrasonic treatment time in the preparation method provided by the present invention is preferably 5 min.

[0144] Example 9

[0145] like Figure 6(a)-Figure 6(c) As shown, this example evaluates the in vitro drug release of the trilobatin-squalene microemulsion composition provided by the present invention.

[0146] Step A1, placing the trilobatin-squalene microemulsion composition provided by the present invention and the reference trilobatin solution in a dialysis bag with a molecular weight of 8000Da, fixing and sealing, and preparing 9 portions in parallel for standby use;

[0147] Step A2: Take 50 mL of each of the following three solutions as dissolution media:

[0148] A. Phosphate buffer containing 10% ethanol, pH 7.4;

[0149] B. Artificial gastric juice prepared according to the 2020 edition of the Chinese Pharmacopoeia, pH 1.2;

[0150] C. Prepare artificial intestinal fluid according to the 2020 edition of the Chinese Pharmacopoeia, with a pH of 6.8.

[0151] Step A3, three parallel tests for each medium. The two groups of sample solutions prepared in step A1 were subjected to trilobatin release experiments at a temperature of 37° C. and a rotation speed of 120 r / min using the three dissolution media obtained in step 2, respectively, and three parallel tests were performed for each medium.

[0152] Step A4, during the trilobatin release experiment, samples were taken at 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, and 48h, 1mL of each time point was sampled, and 1mL of the corresponding dissolution medium was immediately added. After the collected samples were filtered with a 0.22um filter head, the dissolution of the drug was detected by high performance liquid chromatography, and a release rate comparison line graph was drawn. The high performance liquid chromatography detection conditions were: chromatographic column: Kromas i l100-5-C18, 4.6×250nm mobile phase ratio of methanol: water = 58%: 42%, detection wavelength: 280nm, flow rate: 1mL / min.

[0153] From the release test results of trilobatin, as shown in Figure 6(a), the cumulative release of the trilobatin solution control group in a phosphate buffer containing 10% ethanol in the first 12 hours was about 70%; while the cumulative release of the trilobatin-squalene microemulsion composition experimental group in the first 12 hours was 25%, and entered a plateau phase, and the cumulative release was still only about 30% after 48 hours, indicating that the trilobatin-squalene microemulsion composition provided by the present invention has an obvious sustained-release effect in a phosphate buffer containing 10% ethanol at a pH close to neutral.

[0154] like Figure 6(b)-Figure 6(c) As shown, in the gastrointestinal fluid, the control group of the trilobatin solution also showed the characteristics of too fast release speed and large release amount, with the cumulative release reaching 30% in 12 hours, while the release rate and release amount of the trilobatin-squalene microemulsion composition experimental group were significantly smaller.

[0155] This example demonstrates that the trilobatin-squalene microemulsion composition provided by the present invention has an obvious sustained-release effect.

[0156] Example 10

[0157] like Figure 7-Figure 8 As shown, this example verifies the stability of the trilobatin-squalene microemulsion composition provided by the present invention.

[0158] Take an equal amount of the trilobatin-squalene microemulsion composition provided by the present invention and place it in two centrifuge tubes. The two centrifuge tubes are sealed and stored at room temperature and 4°C for 200 days, and samples are taken at 1, 2, 3, 5, 7, 10, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, and 180 days, respectively, and observe whether the appearance of the two groups of trilobatin-squalene microemulsion compositions has precipitation or stratification, and measure whether the particle size and PDI have changed. The observation results show that the trilobatin-squalene microemulsion composition did not show precipitation or stratification when it was left standing for 200 days at room temperature and 4°C; Figure 7-Figure 8 As shown in the two groups of continuous monitoring data, the particle size and PDI of the trilobatin-squalene microemulsion composition did not change significantly when stored for a long time at room temperature and 4°C. This example verifies that the trilobatin-squalene microemulsion composition provided by the present invention has high stability.

[0159] Embodiment 11

[0160] like Figure 9-14 As shown, this example verifies the improving effect of the trilobatin-squalene microemulsion composition provided by the present invention on cerebral ischemia / reperfusion injury.

[0161] Step B1, 40 SPF SD male rats weighing 220-250 g were taken and divided into groups using a simple random method: a normal group, a model group, a model + trilobatin group, a model + squalene group, and a model + trilobatin-squalene microemulsion combination group;

[0162] Step B2, establish a rat focal cerebral ischemia / reperfusion injury model by thread embolization method. Each experimental group was gavaged at 4h, 16h, 28h, 40h, and 52h after modeling, and the gavage reagents of each experimental group are shown in Table 6 below:

[0163] Table 6 Details of intragastric administration reagents for each experimental group in Example 11

[0164]

[0165] After intragastric administration of the reagent, the rats were fed normally for 3 days, and the neurological function was scored using the Longa 5-point scoring standard. The scoring standard is shown in Table 7 below:

[0166] Table 7 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] The neurological function scores of rats in each group were as follows Fig. 9 As shown, compared with the model group, squalene group *p<0.05, trilobatin group **p<0.01, trilobatin-squalene microemulsion combination group ****p<0.0001, the neurological function scores of rats treated with trilobatin-squalene microemulsion combination by gavage were significantly lower than those in other experimental groups.

[0169] Step B3, take some SD rats after the neurological function scoring experiment, use 2% sodium pentobarbital anesthetic solution to anesthetize the SD rats by intraperitoneal injection, and after anesthesia, quickly peel off the SD rat brain tissue on ice, fix it in a -20℃ refrigerator for 20 minutes, slice it, put it in 2,3,5-triphenyltetrazolium chloride stain solution, react at 37℃ 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℃ refrigerator for 48 hours, and then take pictures. The observation results are as follows Fig.10 As shown, the volume of cerebral infarction in rats in the experimental group gavaged with the trilobatin-squalene microemulsion composition was significantly reduced compared with other experimental groups.

[0170] Step B4, use a blade to cut and separate the infarct volume and the non-infarct volume, and calculate the cerebral infarction volume. The calculation formula is as follows:

[0171] Percentage of cerebral infarction volume = (cerebral infarction volume / whole brain tissue) × 100%.

[0172] The calculation results of the percentage of cerebral infarction volume in rats in each experimental group are as follows Fig.11 As shown, ****p<0.0001, model group compared with normal group; **p<0.01, model group compared with trilobatin-squalene microemulsion composition. The cerebral infarction rate of rats in the trilobatin-squalene microemulsion composition experimental group was significantly lower than that in other experimental groups.

[0173] Step B5, take blood from the orbital vein of some SD rats after the neurological function scoring experiment in step B2, 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 method for determining the inflammatory factors in the serum is as follows:

[0174] 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 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. 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 50uL 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.

[0175] The results of the determination of the level of inflammatory factor tumor necrosis factor-α in the serum of SD rats treated with the trilobatin-squalene microemulsion composition are as follows Fig.12 As shown, *p<0.05, model group compared with normal group; *p<0.05, model group compared with trilobatin group; ****p<0.0001, model group compared with combination microemulsion; The results of serum interleukin-1β level determination of SD rats treated with trilobatin-squalene microemulsion composition are shown as follows Fig.13 As shown: *p<0.05, model group compared with normal group; **p<0.01, model group compared with microemulsion of the composition; The results of the determination of the level of interleukin-6 in the serum of SD rats treated with the trilobatin-squalene microemulsion composition are shown as follows Fig.14 As shown: *p<0.05, model group compared with normal group; ***p<0.001, model group compared with microemulsion of the composition. Obviously, the inflammatory factors tumor necrosis factor-α, interleukin-1β, and interleukin-6 in rats treated with trilobatin-squalene microemulsion composition were significantly reduced compared with other experimental groups, proving that it has a significant effect on alleviating reperfusion injury after cerebral ischemia.

[0176] In summary, this example proves through three groups of experimental data that the trilobatin-squalene microemulsion composition provided by the present invention has a significant improvement effect on cerebral ischemia / reperfusion injury.

[0177] Example 12

[0178] like Fig.15 As shown, this example verifies the distribution of the trilobatin-squalene microemulsion composition in vivo.

[0179] SD rats were randomly divided into groups and gavaged with nanoemulsions loaded with DiD. The rats were killed 1.5h and 3h after administration of the trilobatin-squalene microemulsion composition. The heart, liver, spleen, lung, kidney, and brain were removed and washed with saline. The tissues were then dried with filter paper and imaged using the Berthold animal in vivo imaging system. The results are shown in Figure 2. Fig.15 As shown, the trilobatin-squalene microemulsion composition has the strongest fluorescence signal in the kidney, indicating that it is mainly metabolized by the kidney; compared with the free drug, the trilobatin-squalene microemulsion composition has an enhanced fluorescence signal in the brain tissue. This example verifies that the trilobatin-squalene microemulsion composition is more specific to brain tissue than other drugs.

[0180] Embodiment 13

[0181] like Figure 16(a)-Figure 16(d) As shown, this example verifies the effect of the trilobatin-squalene microemulsion composition provided by the present invention on liver and kidney functions.

[0182] Step C1, after adaptive feeding for one week, SPF grade SD male rats were divided into a normal group and a trilobatin-squalene microemulsion composition group (n=3) by simple random method, with a body weight of 220-250 g, and intragastrically administered for three consecutive days, twice a day;

[0183] Step C2, blood was collected from the abdominal aorta, and after standing for 3 hours, the blood was centrifuged at a speed of 12000r / min and a temperature of 4°C for 20 minutes, and the upper serum was collected. The aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, and creatinine in the serum were determined by enzyme-linked immunosorbent assay. The specific determination method is as follows:

[0184] 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 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. 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°C 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°C in the dark for 15 minutes. Add 50uL 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.

[0185] The results of the test are as follows Figure 16(a)-Figure 16(d) As shown, ns: no statistically significant difference, the normal group and the trilobatin-squalene microemulsion composition group were compared. Compared with the normal group, there was no significant difference in the aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine content in the serum of rats treated with the trilobatin-squalene microemulsion composition, indicating that the trilobatin-squalene microemulsion composition provided by the present invention has good safety for both liver and kidney.

[0186] Embodiment 14

[0187] like Figure 17(a)-Figure 18 As shown, this example verifies the biological safety of the trilobatin-squalene microemulsion composition.

[0188] Step D1, after adaptive feeding for one week, SPF SD rats were randomly divided into 3 groups (n=3), and intragastric administration of physiological saline, trilobatin solution and trilobatin-squalene microemulsion composition was performed at a dosage of 5 mg / kg for 3 consecutive days;

[0189] Step D2, on the 5th day, the rats were killed, blood was collected from the abdominal aorta, and red blood cells, white blood cells, platelets, lymphocytes, neutrophils, and monocytes were detected using a veterinary fully automatic blood cell analyzer;

[0190] Step D3, the heart, liver, spleen, lung, kidney and intestine of each group of rats were taken, fixed with 4% paraformaldehyde solution, stained with H&E, and analyzed under an inverted fluorescence microscope.

[0191] Comparison of blood test results among the groups Figure 17(a)-Figure 17(f) As shown, the blood cell test results of rats after intragastric administration of the trilobatin-squalene microemulsion composition were all within the normal range; Fig.18As shown, the H&E staining results showed that there was no obvious lesion in each organ. This test result verifies that the trilobatin-squalene microemulsion composition has good biological safety.

[0192] In summary, the trilobatin-squalene microemulsion composition provided by the present invention can effectively improve the distribution of trilobatin in brain tissue, reduce the volume of cerebral infarction caused by cerebral ischemia or reperfusion injury, improve neurological function scores, and reduce the level of inflammatory factors in the body; its own stability and sustained release effect in related solutions are good, it has good biosafety, and has broad application and market prospects. On the other hand, the preparation method provided by the present invention has simple process and mild conditions, is conducive to large-scale production, and is worthy of wide promotion.

[0193] 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 composition containing trilobatin, characterized in that: The composition comprises trilobatin, squalene, an oil phase and an emulsifier; The mass fractions of the components in the composition are 0.1-5% of trilobatin, 1.3-7% of squalene, 4-10% of oil phase, and 1-3% of emulsifier; The composition is a microemulsion.

2. The composition according to claim 1, characterized in that The composition also includes an emulsifier; The mass fraction of the auxiliary emulsifier in the composition is 0.1-2%.

3. The composition according to claim 1, characterized in that The oil phase is a mixture of one or more of polyoxyethylene hydrogenated castor oil, palm oil, olive oil, soybean oil, triolein, glycerol, and isopropyl myristate.

4. The composition according to claim 1, characterized in that The emulsifier is a mixture of one or more of phospholipids, soybean lecithin, egg yolk lecithin, polyoxyethylene ether, Tween and Span.

5. The composition according to claim 2, characterized in that The auxiliary emulsifier is a mixture of one or more of Tween-80 and polyethylene glycol.

6. A method for preparing the composition according to any one of claims 1 to 5, characterized in that: The specific steps are as follows: Step 1, add 0.1-5 parts of trilobatin, 1.3-7 parts of squalene, 4-10 parts of oil phase, and 1-3 parts of emulsifier into a reaction container, then add an organic solvent, and stir to obtain a mixture solution; Step 2, adding 0.1-2 parts of a co-emulsifier into water to prepare a co-emulsifier solution; under continuous stirring, adding the co-emulsifier solution dropwise into the mixture solution, stirring to obtain colostrum; Step 3, subjecting the colostrum to ultrasonic treatment, and removing the organic solvent by rotary evaporation to obtain a trilobatin-squalene microemulsion composition; The above components are all calculated by mass.

7. The preparation method according to claim 6, characterized in that: The oil phase in step 1 is a mixture of one or more of polyoxyethylene hydrogenated castor oil, palm oil, olive oil, 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 in step 2 is a mixture of one or more of Tween-80, polyethylene glycol and sodium alginate.

8. The preparation method according to claim 6, characterized in that: The power of the ultrasonic treatment is set to 32.5-487.5 W; the time is 5-20 min.

9. A method for improving brain damage during cerebral ischemia-reperfusion, characterized in that: The method adopts the composition according to any one of claims 1 to 5 or the trilobatin-squalene microemulsion composition prepared according to the method according to any one of claims 6 to 8.

10. The method according to claim 9, characterized in that The dosage form of the composition is selected from oral dosage form and injection dosage form.

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