CN113813229A

CN113813229A - Isoliquiritigenin self-microemulsion, preparation method and application thereof in EOE model mice

Info

Publication number: CN113813229A
Application number: CN202111182098.3A
Authority: CN
Inventors: 曹明卓; 高勇; 王泽茜; 陈晓辉; 王媛; 史艳梅
Original assignee: Henan University of Traditional Chinese Medicine HUTCM
Current assignee: Henan University of Traditional Chinese Medicine HUTCM
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2021-12-21
Anticipated expiration: 2041-10-11
Also published as: CN113813229B

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an isoliquiritigenin self-microemulsion, a preparation method and application thereof, and application thereof in an EOE model mouse, wherein the isoliquiritigenin self-microemulsion comprises a surfactant, a cosurfactant, an oil phase and isoliquiritigenin, the surfactant is selected from Tween 80 and polyoxyethylene castor oil, the mass ratio of the surfactant to the surfactant is 7 (2-4), the cosurfactant is PEG400 and 1, 2-propylene glycol, the mass ratio of the cosurfactant to the surfactant is 1 (0.5-1.5), the oil phase is selected from ethyl oleate, and the mass ratio of the oil phase, the surfactant and the cosurfactant is as follows: (2-4): 5-7):1, the isoliquiritigenin reaches a saturation state in the isoliquiritigenin self-microemulsion, and the isoliquiritigenin self-microemulsion is used for treating eosinophilic esophagitis and is applied to an EOE model mouse. The solubility of isoliquiritigenin in the isoliquiritigenin self-microemulsion is obviously improved, and the preparation method does not change the physical and chemical properties of isoliquiritigenin, is simple and is convenient to popularize.

Description

Isoliquiritigenin self-microemulsion, preparation method and application thereof in EOE model mice

Technical Field

The invention relates to the technical field of medicines, in particular to isoliquiritigenin self-microemulsion, a preparation method, application and application thereof in an EOE model mouse.

Background

Eosinophilic esophagitis (eophagitis, abbreviated as EOE) is a chronic inflammatory disease associated with food allergy, and the main pathology is characterized by infiltration of the esophageal mucosa with a large number of Eosinophilic granulocytes (Eosinophil, abbreviated as EOS). The clinical manifestations of adult patients are mainly dysphagia, esophageal stenosis, food incarceration and reflux-like symptoms; children are primarily characterized by food refusal and malnutrition. EOE is a global disease, but is not well-balanced in regional distribution, with a large number of cases seen in north america and south america, eastern and western europe, and australia, and a relatively small number of cases in asia (including the middle east). The prevalence rate of the EOE is reported to be about (5-50)/10 ten thousand in the prior literature, but the prevalence rate of the EOE is in a remarkably increasing trend in recent 20 years, and in the worldwide research report, the local prevalence rate continuously rises and reaches 1: 1000. EOE was first reported in 1978 to be a relatively new disease that is susceptible to missed and misdiagnosis, particularly in developing countries, due to a lack of adequate knowledge of the disease. Indeed, EOE is now becoming an increasingly serious public health hazard not only in western countries, but also in asia.

Eosinophilic esophagitis can not be cured radically so far, and the aim of the treatment is to relieve symptoms, prevent EOE complications and improve the life quality of patients; at present, topical/swallow corticosteroid is selected as the first-line medicament for clinically treating EOE, and although the treatment mode can effectively control the related inflammation of the EOE to a certain extent, the treatment mode has obvious adverse reactions after long-term use, such as oropharyngeal/esophageal candidiasis, abnormal bone density, glaucoma, hyperglycemia, other antagonism and the like. There are currently no FDA-approved drugs for EOE treatment and there is a lack of specific drug formulations for the control of esophageal inflammation.

Disclosure of Invention

In order to solve the technical problems, the invention provides isoliquiritigenin self-microemulsion, a preparation method, application and application thereof in an EOE model mouse.

The technical scheme adopted by the invention is as follows: the invention provides an isoliquiritigenin self-microemulsion which comprises a surfactant, a cosurfactant, an oil phase and isoliquiritigenin, wherein the surfactant is selected from tween 80 and polyoxyethylene castor oil, the mass ratio of the surfactant to the surfactant is 7 (2-4), the cosurfactant is PEG400 and 1, 2-propylene glycol, the mass ratio of the cosurfactant to the surfactant is 1 (0.5-1.5), the oil phase is selected from ethyl oleate, and the mass ratio of the oil phase to the surfactant to the cosurfactant is as follows: (2-4): 5-7):1, wherein the isoliquiritigenin reaches a saturated state in the isoliquiritigenin self-microemulsion.

Preferably, the mass ratio of the oil phase, the surfactant and the cosurfactant is as follows: 3:6:1, the mass ratio of the Tween 80 to the polyoxyethylene castor oil is 7:3, and the mass ratio of the PEG400 to the 1, 2-propylene glycol is 1: 1.

The second aspect of the invention provides a preparation method of the isoliquiritigenin self-microemulsion, which at least comprises the following steps:

s1: preparing a blank nanoemulsion;

s2: adding isoliquiritigenin into the blank nanoemulsion prepared in the step S1 and processing to obtain isoliquiritigenin self-microemulsion.

Preferably, step S1 specifically includes the following steps: (a) adding two cosurfactants into a No. 1 EP tube according to the mass ratio, simultaneously adding two surfactants into a No. 2 EP tube according to the mass ratio, and placing the No. 1 and No. 2 EP tubes in an ultrasonic oscillator to oscillate for 10-20 min; (b) adding a certain mass of oil phase into the No. 3 EP tube, and according to the mass ratio, absorbing a certain mass of surfactant from the No. 2 EP tube and absorbing a certain mass of cosurfactant from the No. 1 EP tube, and adding the cosurfactant into the No. 3 EP tube; (c) placing No. 3 EP tube in ultrasonic oscillator, and oscillating for 10-20min to obtain blank nanoemulsion, and storing at 4 deg.C.

Preferably, step S2 specifically includes the following steps: (1) gradually adding isoliquiritigenin into No. 3 EP tube, and subjecting No. 3 EP tube to ultrasonic oscillation treatment until micro precipitation appears at the bottom of No. 3 EP tube and no change is generated after ultrasonic oscillation treatment for 25-35min, so that the isoliquiritigenin reaches supersaturation state; (2) placing the No. 3 EP tube subjected to the oscillation treatment in a constant-temperature shaking table for 24 hours; and (3) placing the No. 3 EP tube treated by the constant-temperature shaking table in a centrifuge for centrifuging for 30min, and quantitatively absorbing supernatant to obtain the isoliquiritigenin self-microemulsion with saturated isoliquiritigenin content.

In a third aspect, the invention provides an application of the isoliquiritigenin self-microemulsion, wherein the microemulsion is used for treating eosinophilic esophagitis.

The fourth aspect of the invention provides application of isoliquiritigenin self-microemulsion in an EOE model mouse, wherein the EOE model is established for the mouse to obtain the EOE model mouse, the day of the test is marked as day 0, the EOE model mouse is subjected to drug gavage treatment on day 21, and the continuous gavage administration is ended until day 56, wherein the drug is isoliquiritigenin self-microemulsion with a certain mass and is dissolved in 10mL of PBS solution, the mass of isoliquiritigenin in the drug is 20mg, and the equivalent dosage of the drug is 10mL/kg according to the weight of the mouse.

Preferably, the method for establishing the infection EOE model of the mouse comprises the following steps: the experiment is started after the BALB/c mice are adaptively raised for 1 week, the day of starting the experiment is recorded as day 0, the mice are smeared with 20mg of sensitization ointment on the left ear of the mice every day on days 1-7 and days 22-28, and the sensitization ointment comprises the following components in parts by weight: 1 part of peanut protein crude extract and 100 parts of calcipotriol ointment; meanwhile, the mice are injected with the sensitizing agent in the abdominal cavity on the 0 th day, the 7 th day, the 14 th day and the 21 st day respectively, the mice are stimulated by the gastric lavage stimulating agent on the 28 th day, the 35 th day, the 42 th day and the 49 th day, and the mice are stimulated by the gastric lavage large dose stimulating agent before being treated on the 56 th day.

Preferably, the sensitizer comprises the following components: mixing the 20mg peanut protein crude extract, 200mg aluminum hydroxide adjuvant and 10mL PBS solution to obtain a sensitizer, wherein when the sensitizer is injected into the abdominal cavity of a mouse, the injection dosage of the sensitizer is 10mL/kg according to the weight of the mouse; the excitant is a peanut protein crude extract of 25mg/mL, the solvent is PBS, and the dosage of the excitant is 10mL/kg according to the weight of the mouse; the large-dose exciting agent is peanut protein crude extract with the concentration of 50mg/mL, the solvent is PBS, and the dosage of the large-dose exciting agent is 10mL/kg according to the weight of the mouse.

Preferably, the preparation method of the peanut crude extract comprises the following steps: (A) degreasing: drying fresh peanut, peeling, pulverizing peanut kernel into powder with a pulverizer, adding precooled acetone, stirring uniformly, soaking at 4 ℃ for 10-18h, and drying acetone to obtain filter residue; (B) and (3) complete degreasing: adding acetone into the obtained filter residue again, stirring at room temperature for 1-2h, standing, observing whether the supernatant is clear, and performing suction filtration to obtain a solid substance if the supernatant is clear; if the supernatant is not clear, repeating the step B until the supernatant is clear; (C) extraction: and C, adding PBS buffer solution into the solid obtained in the step B, extracting at 4 ℃ for 18-24h, centrifuging for 20min at 1500r/min, taking the middle layer, repeatedly centrifuging, taking supernatant, obtaining the supernatant, namely the crude peanut protein extracting solution, and freeze-drying the crude peanut protein extracting solution and storing at-20 ℃.

Compared with the prior art, the invention has the following advantages: (1) the invention provides an isoliquiritigenin self-microemulsion and a preparation method thereof, the solubility of isoliquiritigenin in the isoliquiritigenin self-microemulsion is obviously improved, and simultaneously the preparation method does not change the physical and chemical properties of isoliquiritigenin, and the preparation method is simple and convenient for popularization; (2) the isoliquiritigenin self-microemulsion is found to be used for treating eosinophilic esophagitis, and the finding makes up for the defect of lack of medicines for treating the eosinophilic esophagitis at present; the invention (3) provides an EOE mouse model for verifying the effect of isoliquiritigenin self-microemulsion on treating esophagitis, the mouse is infected with eosinophilic esophagitis through the mouse model, the EOE mouse model overcomes the defects of in vitro tests, the isoliquiritigenin self-microemulsion is adopted for treating the mouse, and the treatment result proves that the isoliquiritigenin has a certain treatment effect on the eosinophilic esophagitis.

Drawings

FIG. 1 is a flow chart of the creation of a mouse model of eosinophilic esophagitis in accordance with the present invention;

FIG. 2 is a comparison of the isoliquiritigenin self-microemulsion and blank nanoemulsion of the present invention;

FIG. 3 is a comparison of an isoliquiritigenin self-microemulsion after being diluted 10 times with water and a blank nanoemulsion according to the present invention;

FIG. 4 is a comparison of isoliquiritigenin self-microemulsion after 100-fold dilution with water and blank nanoemulsion according to the present invention;

FIG. 5 is a schematic representation of infrared spectra of isoliquiritigenin, blank nanoemulsion and isoliquiritigenin self-microemulsion of the present invention;

FIG. 6 is a schematic diagram showing in vitro release curves of isoliquiritigenin suspension (ILQ) and isoliquiritigenin self-microemulsion (ILQ-SMEDDS) in the present invention;

FIG. 7 is a schematic diagram showing the body temperature changes of the mice in each group within 40min after the last challenge;

FIG. 8 is a graph showing the allergic index profile of each group of mice after the last challenge in accordance with the present invention;

FIG. 9 is a graph showing the change in body weight of groups of mice over the entire duration of the experiment according to the present invention;

FIG. 10 is a graphical representation of spleen and lung indices of various groups of mice at the end of treatment according to the invention;

FIG. 11 is a graph showing the levels of TNF- α at the end of treatment in the sera of various groups of mice in accordance with the invention;

FIG. 12 is a graph showing IL-4 levels at the end of treatment in the sera of groups of mice in the present invention;

FIG. 13 is a graph showing IL-5 levels at the end of treatment in the sera of groups of mice in the present invention;

FIG. 14 is a graph showing the levels of Peanout-S-IgE in the sera of groups of mice at the end of treatment in accordance with the present invention;

FIG. 15 is a schematic structural view of the esophageal and small intestinal tissues at the end of treatment in groups of mice of the present invention;

FIG. 16 is a graphical representation of TGF- β 1 levels in esophageal tissue at the end of treatment for groups of mice in the invention.

Detailed Description

The invention provides an isoliquiritigenin self-microemulsion which comprises a surfactant, a cosurfactant, an oil phase and isoliquiritigenin, wherein the surfactant is selected from tween 80 and polyoxyethylene castor oil, the mass ratio of the surfactant to the surfactant is 7 (2-4), the cosurfactant is PEG400 and 1, 2-propylene glycol, the mass ratio of the cosurfactant to the surfactant is 1 (0.5-1.5), the oil phase is selected from ethyl oleate, and the mass ratio of the oil phase to the surfactant to the cosurfactant is as follows: (2-4): 5-7):1, wherein the isoliquiritigenin reaches a saturated state in the isoliquiritigenin self-microemulsion.

The mass ratio of the oil phase, the surfactant and the cosurfactant is preferably as follows: the mass ratio of 3:6:1, tween 80 and polyoxyethylated castor oil is more preferably 7:3, and the mass ratio of PEG400 and 1, 2-propanediol is more preferably 1: 1.

s1: preparing a blank nanoemulsion; the method specifically comprises the following steps: (a) adding two cosurfactants into a No. 1 EP tube according to the mass ratio, simultaneously adding two surfactants into a No. 2 EP tube according to the mass ratio, and placing the No. 1 and No. 2 EP tubes in an ultrasonic oscillator to oscillate for 10-20 min; (b) adding a certain mass of oil phase into the No. 3 EP tube, and according to the mass ratio, absorbing a certain mass of surfactant from the No. 2 EP tube and absorbing a certain mass of cosurfactant from the No. 1 EP tube, and adding the cosurfactant into the No. 3 EP tube; (c) placing No. 3 EP tube in ultrasonic oscillator, and oscillating for 10-20min to obtain blank nanoemulsion, and storing at 4 deg.C.

S2: adding isoliquiritigenin into the blank nanoemulsion prepared in the step S1 and processing to obtain isoliquiritigenin self-microemulsion; the method specifically comprises the following steps: (1) gradually adding isoliquiritigenin into No. 3 EP tube, and subjecting No. 3 EP tube to ultrasonic oscillation treatment until micro precipitation appears at the bottom of No. 3 EP tube and no change is generated after ultrasonic oscillation treatment for 25-35min, so that the isoliquiritigenin reaches supersaturation state; (2) placing the No. 3 EP tube after the oscillation treatment in a constant temperature shaking table for 24h, wherein the temperature of the constant temperature shaking table is 37 ℃, and the rotating speed is 100 rmp; (3) placing the EP tube No. 3 after being treated by the constant temperature shaking table in a centrifuge for centrifuging for 30min, wherein the rotating speed of the centrifuge is 8000rmp, and quantitatively absorbing the supernatant to obtain the isoliquiritigenin self-microemulsion with saturated isoliquiritigenin content.

Example 1

4.2g of Tween 80, 1.8g of polyoxyethylene castor oil, 0.5g of PEG400, 0.5g of 1, 2-propylene glycol, 3g of ethyl oleate and 1.8g of isoliquiritigenin, wherein a small amount of precipitate still exists at the bottom after ultrasonic oscillation treatment.

The preparation method comprises the following steps: s1: preparing a blank nanoemulsion; the method specifically comprises the following steps: (a) adding two cosurfactants into a No. 1 EP tube according to the mass ratio, simultaneously adding two surfactants into a No. 2 EP tube according to the mass ratio, and placing the No. 1 and the No. 2 EP tubes in an ultrasonic oscillator to oscillate for 15 min; (b) adding a certain mass of oil phase into the No. 3 EP tube, and according to the mass ratio, absorbing a certain mass of surfactant from the No. 2 EP tube and absorbing a certain mass of cosurfactant from the No. 1 EP tube, and adding the cosurfactant into the No. 3 EP tube; (c) placing No. 3 EP tube in an ultrasonic oscillator, and oscillating for 15min to obtain blank nanoemulsion, and storing the blank nanoemulsion at 4 deg.C.

S2: adding isoliquiritigenin into the blank nanoemulsion prepared in the step S1 and processing to obtain isoliquiritigenin self-microemulsion; the method specifically comprises the following steps: (1) gradually adding isoliquiritigenin into the No. 3 EP tube, and performing ultrasonic oscillation treatment on the No. 3 EP tube until trace precipitation appears at the bottom of the No. 3 EP tube and no change is generated after 30min of ultrasonic oscillation treatment, so that the isoliquiritigenin reaches a supersaturated state; (2) placing the No. 3 EP tube after the oscillation treatment in a constant temperature shaking table for 24h, wherein the temperature of the constant temperature shaking table is 37 ℃, and the rotating speed is 100 rmp; (3) placing the EP tube No. 3 after being treated by the constant temperature shaking table in a centrifuge for centrifuging for 30min, wherein the rotating speed of the centrifuge is 8000rmp, and quantitatively absorbing the supernatant to obtain the isoliquiritigenin self-microemulsion with saturated isoliquiritigenin content.

Example 2

5.83g of Tween 80, 1.67g of polyoxyethylene castor oil, 1g of PEG400, 0.5g of 1, 2-propylene glycol, 6g of ethyl oleate and 2.65g of isoliquiritigenin, wherein a small amount of precipitate still exists at the bottom after ultrasonic oscillation treatment; the preparation method is the same as that of example 1.

Example 3

Tween 80 of 7.95g, polyoxyethylene castor oil of 4.55g, PEG400 of 1g, 1, 2-propylene glycol of 1.5g, ethyl oleate of 5g and isoliquiritigenin of 3.5g, wherein a small amount of precipitate still exists at the bottom after ultrasonic oscillation treatment; the preparation method is the same as that of example 1.

Example 4

4.9g of Tween 80, 2.1g of polyoxyethylene castor oil, 0.5g of PEG400, 0.5g of 1, 2-propylene glycol, 4g of ethyl oleate and 2.12g of isoliquiritigenin, wherein a small amount of precipitate still exists at the bottom after ultrasonic oscillation treatment; the preparation method is the same as that of example 1.

The third invention provides application of the isoliquiritigenin self-microemulsion in an EOE model mouse, wherein the EOE model is established for the mouse to obtain the EOE model mouse, the day of the test is marked as day 0, the EOE model mouse is subjected to drug gavage treatment on day 21, the gavage administration is carried out for 5 weeks continuously and every day until day 56, the drug is obtained, the isoliquiritigenin self-microemulsion with a certain mass is dissolved in 10mL of PBS (phosphate buffer solution), the mass of the isoliquiritigenin in the drug is 20mg, and the dosage of the drug is 10mL/kg according to the weight of the mouse.

The method for establishing the mouse infection EOE model comprises the following steps: as shown in figure 1, the experiment is started after the BALB/c mice are adaptively raised for 1 week, the day of the start experiment is marked as day 0, 20mg of sensitization ointment (1 part of peanut protein crude extract and 100 parts of calcipotriol ointment) is smeared on the left ear ears of the mice on days 1-7 and 21-28, meanwhile, the mice are injected with sensitizing agent (20mg of peanut protein crude extract, 200mg of aluminum hydroxide adjuvant and 10mLPBS solution, wherein the concentration of the PBS solution is 0.01mol/L and the injection dosage is 10mL/kg) intraperitoneally on

days

0, 7, 14 and 21 respectively, the mice are stimulated with gastric lavage stimulating agent on

days

28, 35, 42 and 49, the stimulating agent is 25mg/mL of peanut protein crude extract, the solvent is PBS by weight of 0.01mol/L, the dosage of the stimulating agent according to the mouse is 10mL/kg, and stimulating the mice with a large-dose gastric lavage stimulating agent before treatment on the 56 th day, wherein the large-dose stimulating agent is peanut protein crude extract (PPE) with the concentration of 50mg/mL, the solvent is PBS with the concentration of 0.01mol/L, and the dosage of the large-dose stimulating agent is 10mL/kg according to the weight of the mice.

In order to show the treatment effect of the isoliquiritigenin self-microemulsion on EOE, during the test, 30 BALB/c mice are adaptively raised for 1 week and then divided into three groups according to a random digital table method, the three groups are respectively marked as a negative control group, a model group and a microemulsion treatment group, 20mg of sensitizing ointment is smeared on the left ear of the mouse on the 1 st to 7 th days and the 21 st to 28 th days, meanwhile, the mouse injects a sensitizing agent into the abdominal cavity of the mouse on the 0 th day, the 7 th day, the 14 th day and the 21 st day, the mouse is stimulated by intragastric gavage on the 28 th day, the 35 th day, the 42 th day and the 49 th day, and the mouse is stimulated by large-dose intragastric gavage before being treated on the 56 th day. The sensitization ointment smeared on the model group and the microemulsion treatment group comprises the following components in parts by weight: 1 part of peanut protein crude extract and 100 parts of calcipotriol ointment; the sensitizers used in the model group and the microemulsion treatment group comprise the following components: 20mg of peanut protein crude extract, 200mg of aluminum hydroxide adjuvant and 10 mg of PBS solution, wherein the concentration of the PBS solution is 0.01mol/L, the sensitizer is obtained by mixing the three solutions, and when the sensitizer is injected into the abdominal cavity of a mouse, the injection dosage of the sensitizer is 10mL/kg according to the weight of the mouse; the excitant used in the gavage excitation of the model group and the microemulsion treatment group is 25mg/mL peanut protein crude extract, the solvent is PBS with the concentration of 0.01mol/L, and the dosage of the excitant is 10mL/kg according to the weight of the mouse; when the model group and the micro-emulsion treatment group are stimulated by large-dose intragastric administration, a large-dose excitant is used, the large-dose excitant is 50mg/mL peanut protein crude extract, a solvent is 0.01mol/L PBS, and the dosage of the large-dose excitant is 10mL/kg according to the weight of a mouse. The sensitization ointment smeared on the mice of the negative control group is calcipotriol ointment without peanut protein crude extract, the dosage of the sensitization ointment is the same as that of the other two groups, and the sensitizing agent, the gastric lavage stimulation and the large-dose gastric lavage stimulation injected by the negative control group are PBS solutions, and the dosage of the medicines is also the same as that of the other two groups.

The mice in the microemulsion treatment group are subjected to intragastric administration every day from day 21, the continuous intragastric administration is finished until day 56, the administered medicine is a mixture of isoliquiritigenin self-microemulsion and 10mL PBS solution, the mass of the isoliquiritigenin in the medicine is 20mg, and the dosage of the medicine is 10mL/kg according to the weight of the mice; negative control group

The placebo is administered to the mice by intragastric administration every day from day 21, and the placebo is administered by continuous intragastric administration until day 56, wherein the placebo is a PBS solution without any solute and is used in an amount of 10 mL/kg; the model groups were not dosed or placebo treated.

The preparation method of the peanut crude extract comprises the following steps: (A) degreasing: drying and peeling fresh peanuts, crushing peanut kernels into powder by using a crusher, adding precooled acetone, uniformly stirring, soaking at 4 ℃, optionally soaking in a refrigerator cold storage chamber for 10-18h, and drying the acetone by using a rotary evaporator in a fume hood to obtain filter residues; (B) and (3) complete degreasing: adding acetone into the obtained filter residue again, stirring at room temperature for 1-2h, standing, observing whether the supernatant is clear, and performing suction filtration to obtain a solid substance if the supernatant is clear; if the supernatant is not clear, repeating the step B until the supernatant is clear; (C) extraction: and C, adding PBS buffer solution into the solid obtained in the step B, extracting at 4 ℃ for 18-24h, centrifuging for 20min at 1500r/min, taking the middle layer, repeatedly centrifuging, taking supernatant, obtaining the supernatant, namely the crude peanut protein extracting solution, and freeze-drying the crude peanut protein extracting solution and storing at-20 ℃.

The isoliquiritigenin self-microemulsion performance characterization comprises the following aspects:

1. determination of concentration of isoliquiritigenin in self-microemulsion

Dissolving a proper amount of isoliquiritigenin in absolute ethyl alcohol, preparing different concentrations, uniformly mixing, detecting absorbance at 370nm to obtain a standard curve of y being 0.125x-0.0152, dissolving a certain weight of isoliquiritigenin in the microemulsion by using the absolute ethyl alcohol, detecting the absorbance at 370nm, calculating the solubility of the isoliquiritigenin according to the dilution times, wherein the drug content of the isoliquiritigenin in the isoliquiritigenin self-microemulsion prepared in example 1 in the test is 12.77% (mass fraction).

2. Appearance of isoliquiritigenin self-microemulsion

As shown in figure 2, blank nano-emulsion and isoliquiritigenin self-microemulsion are respectively placed in a strain bottle for observation, as shown in figure 3, blank nano-emulsion and isoliquiritigenin self-microemulsion which are diluted by 10 times by pure water are respectively placed in a strain bottle for observation, as shown in figure 4, blank nano-emulsion and isoliquiritigenin self-microemulsion which are diluted by 100 times by pure water are respectively placed in a strain bottle for observation, and the result shows that the isoliquiritigenin self-microemulsion is transparent and clear, the blank nano-emulsion diluted by 10 times by pure water is transparent and has blue light, the blank nano-emulsion diluted by 100 times by pure water is slightly transparent and has weak blue light, and the isoliquiritigenin self-microemulsion diluted by 10 times and 100 times is both clear and transparent, so the isoliquiritigenin self-microemulsion is a stable self-microemulsion system.

3. Infrared characterization of isoliquiritigenin self-microemulsion

The potassium bromide tabletting method is adopted, and the infrared spectrum of isoliquiritigenin standard, blank nanoemulsion and isoliquiritigenin self-microemulsion is recorded on a Fourier transform infrared spectrometer, and the spectrum is shown in figure 5.

In Fourier transform infrared spectrum at 3426cm^-1For, 2858cm^-1、1606cm^-1And 1363cm^-1The characteristic absorption peaks show obvious changes, and the changes of the intensity of the absorption peaks indicate that the isoliquiritigenin in the isoliquiritigenin self-microemulsion generates new interactions with media, and the interactions enhance the characteristic peaks of the isoliquiritigenin but do not generate new bonds.

4. Stability of isoliquiritigenin self-microemulsion

The stability of the isoliquiritigenin self-microemulsion was studied under different storage time, temperature and different pH values, and the changes of particle size, polydispersity index (PDI) and zeta potential were examined, and the results are shown in tables 1 and 2. The isoliquiritigenin self microemulsion was stored in a vial and stored at 37 ℃ and 4 ℃ for 2 months, respectively, diluted with 100-fold ultrapure water for predetermined times (0 month, 1 month, and 2 months), and then its particle diameter, polydispersity index (PDI), and ZETA potential (ZETA potential) were measured. To evaluate whether the isoliquiritigenin self-microemulsion is destroyed in the gastrointestinal tract, freshly prepared isoliquiritigenin self-microemulsion was diluted to a predetermined degree with simulated gastric juice (hydrochloric acid having pH of 1.2) and intestinal juice (PBS having pH of 6.8), and the size, polydispersity index and zeta potential of the diluted globular emulsion were measured, and the results are shown in table 3.

TABLE 1 stability of isoliquiritigenin self-microemulsions at room temperature for different storage times

Items	Particle size (nm)	PDI	Zeta potential (mV)
				0 month	27.25	0.186	-10.97
One month	29.30	0.219	-8.35
				Two months old	241.48	0.237	-4.41

TABLE 2 stability of isoliquiritigenin self-microemulsions at different storage temperatures and storage times

Items	Particle size (nm)	PDI	Zeta potential (mV)
				4 ℃ per month	28.89	0.206	-8.34
37 ℃ for one month	29.30	0.211	-8.35
				4 ℃ for two months	140.41	0.224	-8.12
37 ℃ for two months	241.48	0.237	-4.41

TABLE 3 Change of isoliquiritigenin self-microemulsion after acid and alkali influence

	Particle size (nm)	PDI	Zeta potential (mV)
				PBS	28.85	0.191	-
HCl	28.33	0.204	-

Although the isoliquiritigenin self-microemulsion is a thermodynamically stable system, the isoliquiritigenin self-microemulsion is likely to be aggregated with the prolonging of time and the rising of temperature, and the pH value is changed from 1.5-6.8 without generating obvious particle size and PDI changes (the shape and the structure are not obviously changed), which shows that the isoliquiritigenin self-microemulsion can bear the pH value change of body fluid of the whole gastrointestinal tract (GIT) and has a stable structure.

5. In vitro release test of isoliquiritigenin self-microemulsion

The in vitro release profiles of isoliquiritigenin suspension and isoliquiritigenin self-microemulsion in two media were determined by dialysis-diffusion method (method of reference and slightly modified). Placing isoliquiritigenin suspension (2 mL of the suspension contains PBS medium, 5mg of isoliquiritigenin and 1% of Tween 80, and the rest is PBS) and isoliquiritigenin self-microemulsion diluent (2 mL of the diluent contains PBS medium and isoliquiritigenin self-microemulsion containing equal amount of isoliquiritigenin, namely 5mg of isoliquiritigenin is also contained in the diluent) in dialysis solution bags with molecular weight cut-off value of 5000Da respectively, and then placing the dialysis solution bags in 250 mL of release medium (hydrochloric acid solution with pH value of 1.2 for the first 2 hours and PBS solution with pH value of 6.8 for the rest 10 hours) in a constant temperature oscillator with 100 rpm/min. The results of measuring the content of isoliquiritigenin in the dialysate by uv-vis spectrophotometry were shown in table 4 and fig. 6, wherein the data in table 4 are the average values of three parallel tests, by taking 2mL of each dialysate and rapidly supplementing an equal volume of release medium at 0, 0.25, 0.5, 0.75, 1,2, 4, 8, and 12 hours.

TABLE 4 in vitro Release degree of Isoliquiritigenin from microemulsion

Time (h)	0	0.25	0.5	0.75	1.0	2.0	4.0	8.0	12.0
										Degree of suspension Release (%)	0	3.89	8.93	21.37	35.49	45.28	50.82	53.36	57.53
Degree of self-microemulsion Release (%)	0	1.77	3.46	8.15	15.71	28.29	48.86	65.04	76.58

As can be seen from table 4 and fig. 6, since the solubility of isoliquiritigenin is very low, 1% tween 80 surfactant was added to the in vitro release solution to dissolve isoliquiritigenin released from the dialysis bag. The release of isoliquiritigenin suspension in hydrochloric acid (simulated gastric fluid) with pH value of 1.2 at 2h reaches 45.28%, and the release of isoliquiritigenin suspension in phosphoric acid buffer (simulated intestinal fluid) with pH value of 6.8 between 2h and 12h is only increased by 12.25%. Compared with the suspension, the isoliquiritigenin self-microemulsion releases relatively slowly in simulated gastric juice (pH is 1.2 hydrochloric acid), which shows that the isoliquiritigenin nano self-microemulsion can effectively avoid the sudden release of isoliquiritigenin in gastric juice; the release amount of the isoliquiritigenin self-microemulsion in a phosphate buffer solution with the pH value of 6.8 is increased by 48.29 percent between 2h and 12h and is far higher than that in a suspension, which shows that the nano self-microemulsion can continuously release the isoliquiritigenin medicament under the same condition, the burst release of the medicament can be effectively avoided, and the blood concentration is relatively stable.

Application of (di) isoliquiritigenin self-microemulsion in EOE model mouse

1. Body weight and apparent sign changes in three groups of mice

After the last large dose challenge, i.e., day 56, behavioral changes, i.e., allergic index, were recorded using blind observation, with the following scoring criteria: asymptomatic score 0; the number of harassing heads and noses is 1 point; diarrhea, edema around eyes and mouth, upright hair, and reduced frequency of activity of 2 min; cyanosis of the mouth and tail and difficulty in breathing for 3 minutes; no or only mild reaction, tremor and spasm after stimulation were divided into 4 points; the death was divided into 5 points. In addition, the change in body temperature within 40min after the mice were challenged was measured with an infrared thermometer and recorded one by one. The results of the body temperature changes of the mice are shown in FIG. 7, and the results of the allergy index are shown in FIG. 8. At the same time, the weight change from the first intragastric challenge to the last intragastric challenge was recorded for three groups of mice, and the results are shown in fig. 9. After the appearance observation was completed, the mice were dissected, and the organ index was calculated by weighing the organs of the mice, and the results are shown in fig. 10.

Allergic reactions are systemic, even life-threatening diseases triggered by mediators released from mast cells and basophils, and can be activated by either allergic (IgE-mediated) or non-allergic (non-IgE-mediated) mechanisms; it is a rapidly developing multisystemic process involving the skin, lung, gastrointestinal and cardiovascular systems. In the clinic, food allergy and eosinophilic esophagitis are often IgE-mediated.

By adopting the PPE-induced EOE mouse model, the typical symptoms and pathological changes of eosinophilic esophagitis can be well simulated, and the typical characteristics of food allergy can be realized. Compared with the negative control group mice, the model group mice show slow action, poor hair luster and loose excrement after the second excitation, wherein the ear parts of the mice show red swelling, red-spot bleeding, blood crusting and even thickening. After the PPE is excited by a large dose, the model mice show different weight loss, and the pathogenesis of the esophagitis can be effectively reversed by isoliquiritigenin self-microemulsion treatment, and the symptoms are greatly improved and relieved.

Fig. 9 shows the weight change of each group of mice during the whole experiment, and the weight of the model group of mice is obviously reduced after the first excitation, the average weight of the model group of mice is already 0.63g lower than the average weight before the experiment, and the weight of the model group of mice is reduced to different degrees after each excitation. By the end of the experiment, the average body weight of the model group mice decreased by 2.71 g. Although the body weight of the mice in the microemulsion group after the isoliquiritigenin self-microemulsion treatment is reduced to different degrees after the excitation, the body weight of the mice in the group is increased by 0.63g in the 4 th excitation compared with the initial test, and the body weight of the mice in the group is reduced by 0.29g in the last excitation compared with the initial test.

FIG. 7 shows the body temperature changes of the mice in each group within 40min after the last challenge, except for the negative control group, the mice in each group all showed different degrees of body temperature decrease within 40min after the challenge, and the body temperature began to rise to some extent at 40 min. Compared with a negative control group, the body temperature of the mice in the model group and the microemulsion group is most obviously reduced at 20 min-30 min, and the significant difference is realized (p is less than 0.01, and p is less than 0.05); the body temperature of the model group mice is most obviously reduced by 2.3 ℃ compared with the basal body temperature; the degree of the obvious reduction of the body temperature of the mice treated by the isoliquiritigenin self-microemulsion is relieved to a certain degree.

Figure 8 shows the results of the allergic index after the last challenge for each group of mice. Mice in the model group and the microemulsion treatment group have allergic manifestations of different degrees of no pronation, upright hair, nose scratching, face scratching, diarrhea, cyanosis and the like after being stimulated; the negative control mice had no such allergic and asthmatic manifestations except occasional nasal scratching. After the last challenge, figure 8 records the allergic behavioral manifestations of each group of mice within 15min (and scores the most severe behavioral manifestations according to a semi-quantitative score), and compared with a negative control group, the mice in the model group have significantly increased allergic symptom scores and have very significant differences (p < 0.001); the anaphylaxis symptoms of the mice are relieved to a certain extent after the isoliquiritigenin self-microemulsion treatment, the anaphylaxis score of the mice in the microemulsion group is obviously lower than that of the mice in the model group, and the mice have statistical difference (p is less than 0.05).

As shown in fig. 10, since eosinophilic esophagitis is an autoimmune disease, the lung coefficient and spleen coefficient of the model mice are significantly increased (p <0.001), the lung index is increased by about 151.5% and the spleen index is increased by 208.0% in comparison with the negative control group, due to the inflammation of the lung and the enlargement of the spleen in the experimental process; compared with a negative control group, the lung coefficient and the spleen coefficient of the mice in the microemulsion group are also increased, but compared with the mice in the model group, the spleen coefficient and the lung coefficient of the mice in the microemulsion group are respectively reduced by 15.3% and 12.0% (p is less than 0.05, and p is less than 0.05), which indicates that the isoliquiritigenin self-microemulsion can improve the inflammation of the spleen and the lung of the mice to a certain extent.

2. Cytokine detection

After the observation of the change of the apparent physical signs is finished, the mice are taken out, the eyeballs are picked up, blood is taken out, the taken blood is centrifuged for 10min in a centrifuge at the rotating speed of 12000rpm/min, and serum is separated. According to the ELISA kit operation instructions, the levels of specific IgE, IL-4, IL-5 and TNF-alpha in the mouse serum are detected.

The pathogenesis of EOE is not completely understood, and most researchers believe that EOE is an immune response mediated by Th2, and the Th2 cytokine products, IL-4, IL-5 and the eosinophil effector, all play key roles in the pathogenesis of EOE. Therefore, we measured the levels of cytokines such as IL-4, IL-5, TNF-alpha, etc., in the serum of each group of mice.

As shown in FIGS. 11-14, compared with the negative control group mice, the serum contents of TNF-alpha, IL-4 and IL-5 in the model group mice are all obviously increased (p is less than 0.01); compared with the model group, the mice in the microemulsion group are treated to reduce the inflammatory factors to different degrees.

TNF- α is the earliest and most important inflammatory mediator in the inflammatory response. As shown in FIG. 11, the serum TNF-alpha concentration of the model group mice is significantly increased to 484.57 ng/mL; the concentration of TNF-alpha in the serum of the micro-emulsion mice is obviously reduced by about 33.3 percent (p is less than 0.05) compared with that of the model mice.

As shown in fig. 12 and fig. 13, IL-4 and IL-5 in the serum of the mice in the microemulsion group were significantly reduced by 56.7% and 39.6%, respectively, compared to the model group, and the reorganization data had very significant statistical differences (p <0.01 ).

IgE is a key immunoglobulin in the pathogenesis of IgE-mediated related allergic diseases. FIG. 14 shows the results of detection of the levels of Peanut-Specific IgE (Peanout-Specific-IgE, Peanout-S-IgE) by ELISA. Compared with a negative control group, the serum of the model group mouse has the characteristics that the Peanut Peanout-S-IgE is obviously increased, the average concentration reaches 369.89ng/mL, and the statistical difference is extremely significant (p is less than 0.0001). The peripheral blood Peanout-S-IgE of the micro-emulsion mice treated by the isoliquiritigenin self-micro-emulsion is reduced to 220.30ng/mL, and compared with the model mice, the value is obviously reduced by about 40 percent (p is less than 0.01).

3. H & E staining for lung histological changes

The mice were sacrificed, esophageal, pulmonary, and intestinal tissues were taken and fixed in 4% paraformaldehyde, paraffin-embedded and sectioned, and hematoxylin-eosin (H & E) staining was performed on pathological specimens, and pathological conditions such as inflammatory infiltration, tissue damage, intestinal epithelial cell villus deformation, and edema in the tissues were observed, and the results are shown in fig. 15.

Pathological changes of the EOE model mouse can be visually evaluated by the pathological section. As shown in A in FIG. 15, the negative control mice had clear esophageal structure, uniform esophageal wall, occasional little inflammatory cell infiltration around the esophageal wall, and no congestion in the esophageal wall. The esophageal tissue structure of a model group mouse is abnormal, the esophageal wall is obviously thickened, the swelling and congestion are serious, and meanwhile, a large amount of inflammatory cell infiltration and eosinophilic granulocyte infiltration are accompanied around the esophagus; compared with a model group, the inflammatory infiltration condition of the esophageal tissue of a micro-emulsion group mouse is obviously reduced, the thickening of the esophageal wall is not obvious, and the swelling condition is also relieved.

As shown in B in FIG. 15, the tissue structure of the small intestine of the mice in the negative control group is clear, and a small amount of inflammatory cell infiltration is occasionally observed in the submucosa, and the structure is not loose. The intestinal tissue structure of the model group mice is abnormal, and the mice are damaged and infiltrated by a large amount of inflammatory cells; the micro-emulsion group treated by the isoliquiritigenin self-micro-emulsion has the advantages that the small intestine tissue pathology is improved, the structural loosening symptom is still existed, and the tissue still has certain damage.

4. Immunohistochemistry

Paraffin sections were subjected to immunohistochemical analysis by adding TGF- β 1 antibody and ii antibody after fixed by deparaffinization, followed by staining of the mounting, the results of which are shown in fig. 16. TGF-beta 1 has the functions of regulating immune response and causing fibrosis, and is the most critical cytokine for mediating tissue inflammation and regulating extracellular matrix deposition. As shown in FIG. 16, the staining of the cells localized to the cytoplasm, was yellowish brown and diffusely distributed as positive expression of TGF-. beta.1. TGF-beta 1 has higher positive expression level in esophageal tissues of mice in a model group and a microemulsion group, and the positive expression of mice in a negative control group is lower.

5. Statistical analysis

Statistical analysis was performed using Prism 8 software. All data are expressed as mean ± standard deviation (mean ± SD). One-way ANOVA (one-way ANOVA) is adopted for comparison among multiple groups, and t test is adopted for comparison between every two groups. Differences of p <0.05 were statistically significant.

The foregoing description is only of the preferred embodiments of the present invention, and it should be noted that various changes and modifications can be made by those skilled in the art without departing from the overall concept of the invention, and these should also be considered as the protection scope of the present invention.

Claims

1. The isoliquiritigenin self-microemulsion is characterized in that: the isoliquiritigenin self-microemulsion comprises a surfactant, a cosurfactant, an oil phase and isoliquiritigenin, wherein the surfactant is selected from Tween 80 and polyoxyethylene castor oil, the mass ratio of the Tween 80 to the polyoxyethylene castor oil is 7 (2-4), the cosurfactant is PEG400 and 1, 2-propylene glycol, the mass ratio of the cosurfactant to the polyoxyethylene castor oil is 1 (0.5-1.5), the oil phase is selected from ethyl oleate, and the mass ratios of the oil phase, the surfactant and the cosurfactant are as follows: (2-4): 5-7):1, wherein the isoliquiritigenin reaches a saturated state in the isoliquiritigenin self-microemulsion.

2. The isoliquiritigenin self-microemulsion according to claim 1, wherein: the oil phase, the surfactant and the cosurfactant are in the following mass ratio: 3:6:1, the mass ratio of the Tween 80 to the polyoxyethylene castor oil is 7:3, and the mass ratio of the PEG400 to the 1, 2-propylene glycol is 1: 1.

3. The method for preparing an isoliquiritigenin self-microemulsion according to any one of claims 1-2, wherein: the method at least comprises the following steps:

s1: preparing a blank nanoemulsion;

4. The method for preparing an isoliquiritigenin self-microemulsion according to claim 3, wherein the method comprises the following steps: step S1 specifically includes the following steps: (a) adding two cosurfactants into a No. 1 EP tube according to the mass ratio, simultaneously adding two surfactants into a No. 2 EP tube according to the mass ratio, and placing the No. 1 and No. 2 EP tubes in an ultrasonic oscillator to oscillate for 10-20 min; (b) adding a certain mass of oil phase into the No. 3 EP tube, and according to the mass ratio, absorbing a certain mass of surfactant from the No. 2 EP tube and absorbing a certain mass of cosurfactant from the No. 1 EP tube, and adding the cosurfactant into the No. 3 EP tube; (c) placing No. 3 EP tube in ultrasonic oscillator, and oscillating for 10-20min to obtain blank nanoemulsion, and storing at 4 deg.C.

5. The method for preparing an isoliquiritigenin self-microemulsion according to claim 4, wherein the method comprises the following steps: step S2 specifically includes the following steps: (1) gradually adding isoliquiritigenin into No. 3 EP tube, and subjecting No. 3 EP tube to ultrasonic oscillation treatment until micro precipitation appears at the bottom of No. 3 EP tube and no change is generated after ultrasonic oscillation treatment for 25-35min, so that the isoliquiritigenin reaches supersaturation state; (2) placing the No. 3 EP tube subjected to the oscillation treatment in a constant-temperature shaking table for 24 hours; (3) placing the EP tube No. 3 treated by the constant temperature shaking table in a centrifuge for centrifuging for 30min, and quantitatively sucking the supernatant to obtain the isoliquiritigenin self-microemulsion with saturated isoliquiritigenin content.

6. Use of an isoliquiritigenin self-microemulsion according to any one of claims 1-5, wherein: the microemulsion can be used for treating eosinophilic esophagitis.

7. Use of an isoliquiritigenin self-microemulsion according to any one of claims 1-6 in an EOE model mouse, wherein: establishing a model of infecting EOE on a mouse to obtain an EOE model mouse, recording the day of starting a test as day 0, carrying out drug gavage treatment on the EOE model mouse on day 21, and continuously carrying out gavage administration until the day 56 is finished, wherein the drug is isoliquiritigenin self-microemulsion with a certain mass and is dissolved in 10mL of PBS solution, the mass of the isoliquiritigenin in the drug is 20mg, and the dosage of the drug is 10mL/kg according to the weight of the mouse.

8. The use of an isoliquiritigenin self-microemulsion according to claim 7 in an EOE model mouse, wherein: the method for establishing the mouse infection EOE model comprises the following steps: the experiment is started after the BALB/c mouse is adaptively raised for 1 week, the day of the starting experiment is recorded as day 0, the mouse coats 20mg of sensitization ointment on the left ear of the mouse on days 1-7 and 21-28, and the sensitization ointment comprises the following components in parts by weight: 1 part of peanut protein crude extract and 100 parts of calcipotriol ointment; meanwhile, the mice are injected with the sensitizing agent in the abdominal cavity on the 0 th day, the 7 th day, the 14 th day and the 21 st day respectively, the mice are stimulated by the gastric lavage stimulating agent on the 28 th day, the 35 th day, the 42 th day and the 49 th day, and the mice are stimulated by the gastric lavage large dose stimulating agent before being treated on the 56 th day.

9. The isoliquiritigenin self-microemulsion according to claim 8, wherein: the sensitizer comprises the following components: mixing 20mg of Peanut Protein crude Extract (PPE), 200mg of aluminum hydroxide (Alum) adjuvant and 10mL of PBS solution to obtain a sensitizer, wherein when the sensitizer is injected into the abdominal cavity of a mouse, the injection dosage of the sensitizer is 10mL/kg according to the weight of the mouse; the excitant is a peanut protein crude extract of 25mg/mL, the solvent is PBS, and the dosage of the excitant is 10mL/kg according to the weight of the mouse; the large-dose exciting agent is peanut protein crude extract with the concentration of 50mg/mL, the solvent is PBS, and the dosage of the large-dose exciting agent is 10mL/kg according to the weight of the mouse.

10. The isoliquiritigenin self-microemulsion according to any one of claims 7-9, wherein: the preparation method of the peanut crude extract comprises the following steps: (A) degreasing: drying fresh peanut, peeling, pulverizing peanut kernel into powder with a pulverizer, adding precooled acetone, stirring uniformly, soaking at 4 ℃ for 10-18h, and drying acetone to obtain filter residue; (B) and (3) complete degreasing: adding acetone into the obtained filter residue again, stirring at room temperature for 1-2h, standing, observing whether the supernatant is clear, and performing suction filtration to obtain a solid substance if the supernatant is clear; if the supernatant is not clear, repeating the step B until the supernatant is clear; (C) extraction: and C, adding PBS buffer solution into the solid obtained in the step B, extracting at 4 ℃ for 18-24h, centrifuging for 20min at 1500r/min, taking the middle layer, repeatedly centrifuging, taking supernatant, obtaining the supernatant, namely the crude peanut protein extracting solution, and freeze-drying the crude peanut protein extracting solution and storing at-20 ℃.