CN113209082A

CN113209082A - Application of food allergy inhibitor and ginkgolide B in preparation of medicine for preventing and/or treating food allergy

Info

Publication number: CN113209082A
Application number: CN202110612683.6A
Authority: CN
Inventors: 麻小娟; 杨晓童; 杨英; 夏娴; 胡斌丽; 李岩
Original assignee: Zunyi Medical University
Current assignee: Zunyi Medical University
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-08-06

Abstract

The invention provides an application of a food allergy inhibitor and ginkgolide B in preparation of a medicine for preventing and/or treating food allergy, and belongs to the technical field of allergy medicines. The invention provides an application of ginkgolide B in preparing a medicament for preventing and/or treating food allergy. The invention takes a mouse model with food allergy as an experimental object, takes a blank control group, a model group and a positive drug group as controls, and respectively measures the allergic symptoms and the physiological indexes of the organism allergic reaction, and the result shows that the ginkgolide B has the functions of improving the allergic symptoms of the mouse model with food allergy and reducing the organism allergic reaction, has the function of inhibiting food allergy, and has the effect basically consistent with that of the positive drug FAHF-2. Therefore, the ginkgolide B can be used for preparing a food allergy inhibitor to achieve the purpose of preventing and/or treating food allergy diseases.

Description

Application of food allergy inhibitor and ginkgolide B in preparation of medicine for preventing and/or treating food allergy

Technical Field

The invention belongs to the technical field of allergy medicines, and particularly relates to an application of a food allergy inhibitor and ginkgolide B in preparation of a medicine for preventing and/or treating food allergy.

Background

Food allergy epidemiological investigations report that the incidence of food allergy has a rapidly increasing trend in recent years. Food allergy causes great trouble to the patient himself and his family members, and is also a great medical burden in China. Therefore, food allergy has become a serious public health problem, and has attracted a wide attention in the world. In the aspect of food allergy treatment, no specific treatment method exists at present, and treatment medicines such as epinephrine, diphenhydramine hydrochloride or cetirizine are generally given according to the severity of allergic reaction of a patient. However, these western medicines have side effects and are only directed to the corresponding symptoms of food allergy, and cannot fundamentally treat immune disorders of patients.

The active ingredients of the traditional Chinese medicine are good choices for treating food allergy, and the compound traditional Chinese medicine with a treatment effect on food allergy comprises Formula-1(FAHF-1), Formula-2(FAHF-2), Formula-3(FAHF-3), CRFC-1 and the like, wherein the FAHF-2 has a good treatment effect and is deeply researched. However, FAHF-2 is a compound traditional Chinese medicine, which has the disadvantage of inconvenient administration, and the effective medicinal component of FAHF-2 is berberine, because the bioavailability of berberine monomer is low, other substances in the prescription are needed to assist the absorption of berberine, which limits the single administration of the berberine^[1]. Therefore, a single traditional Chinese medicine or a traditional Chinese medicine active ingredient has great market prospect for effectively treating food allergy.

Ginkgo biloba is a specialty plant in China. The planting area of the gingko in China accounts for about 70% of the total world amount, and a large number of natural gingko communities, semi-natural gingko communities and artificial communities exist in China, so that the research related to the gingko in China occupies geographical and resource advantages. Folium Ginkgo, fruit, seed coat, and kernel can be used as medicine, and bilobalide B (GB) is contained in the leaf extractIs one of very important active ingredients, the GB reported at present has the effects of resisting oxidation, delaying senility and the like, and is commonly used for treating pancreatitis, thrombus and cardiovascular diseases clinically^[2]. However, no report is found on whether GB has a therapeutic effect on food allergy.

Reference documents:

[1]Yang N,Srivastava K,Song Y,et al.Berberine as a chemical and pharmacokinetic marker ofthe butanol-extracted FoodAllergy Herbal Formula-2.Int Immunopharmacol,2017,45:120-127.

[2]Chu X,Ci X,He J,et al.A Novel Anti-Inflammatory Role for Ginkgolide B in Asthma via Inhibition ofthe ERK/MApK Signaling pathway[J].Molecules,2011,16(9):7634-7648.

disclosure of Invention

In view of the above, the present invention aims to provide a new use of ginkgolide B, i.e., an application of ginkgolide B in preparing a medicament for preventing and/or treating food allergy.

The invention also aims to provide a food allergy inhibitor, which enriches the types of the existing food allergy inhibitors and provides a new idea for food allergy treatment.

The invention provides an application of ginkgolide B in preparing a medicament for preventing and/or treating food allergy, wherein the structure of ginkgolide B is shown as formula I:

preferably, the application of the ginkgolide B and the food allergy inhibitor in preparing the medicine for preventing and/or treating food allergy.

Preferably, the food allergy inhibitor comprises a traditional Chinese medicine compound food allergy inhibitor and a western medicine food allergy inhibitor.

Preferably, the traditional Chinese medicine compound food allergy inhibitor comprises one or more of the following components: FAHF-1, FAHF-2, FAHF-3 and CRFC-1.

Preferably, the western medicine food allergy inhibitor comprises at least one of the following components: epinephrine, levocetirizine hydrochloride, fexofenadine, diphenhydramine hydrochloride, and topical calamine lotion.

Preferably, the food comprises eggs, dairy products, dried fruits, seafood, beef or sesame, soy and related products.

The invention provides a food allergy inhibitor, which takes ginkgolide B as a medicinal active ingredient.

Preferably, the active ingredients of the medicine also comprise a traditional Chinese medicine compound food allergy inhibitor.

The invention provides an application of ginkgolide B in preparing a medicament for preventing and/or treating food allergy. The food allergy test method takes a mouse model with food allergy as an experimental object, low-dose and high-dose ginkgolide B is administered, FAHF-2 is taken as a positive drug control, a blank control mouse group and an allergic model group are simultaneously arranged, the influence of drug treatment on the food allergy mouse model is evaluated respectively from the aspects of allergic symptoms, mouse intestinal morphology, specific antibody IgE, IgG and IgG subtype levels in serum, mouse spleen cytokine levels, T cell subgroup quantity and the like, and the experimental result shows that the mouse allergic symptoms of GB stem prognosis are obviously improved, and the mouse allergic symptom score is the same as that of the positive drug FAHF-2 group; GB low-dose administration can obviously improve the shape of the small intestine and has the effect close to that of an FAHF-2 intervention group; the administration of low dose of GB makes the level of IgE antibody, IgG and IgG subtype in a mouse model obviously lower than that in a model group, and shows similar effect with an FAHF-2 intervention group; the low dose of GB administration significantly reduced the levels of IL-4, IL-13, IL-17A and IFN-gamma in the mouse model indicating effective reduction of allergic reactions; compared with the mice in the sensitized group, the number of Th2 and Th17 in the Chinese medicine GB0.1 mg intervention group is obviously reduced, the ratio of Foxp3+ Treg is obviously increased, and the number change of each T cell subtype shows similarity compared with the FAHF-2 group. In conclusion, GB has good inhibition effect on food allergy mouse model symptoms and anaphylactic reaction, and is suitable for preparing the medicine for preventing and/or treating food allergy diseases.

The application provided by the invention further limits the application of the ginkgolide B and other food allergy inhibitors in the preparation of the medicine for preventing and/or treating food allergy diseases. The scheme of the invention can widen the active ingredients for inhibiting food allergy in the prior art, and provides a new idea for developing new specific medicines.

Drawings

FIG. 1 is a histogram of IgE levels in mouse serum before and after modeling; wherein BLANK: blank control group; m: a sensitization model group;

FIG. 2 is a bar graph of allergic symptom scores of mice of different treatment groups; wherein BLANK: blank control group; m: a sensitization model group; GBL: GB0.1 mg/d low dose group; GBH: GB0.8 mg/d high dose group; FAHF-2: a positive control group;

FIG. 3 is a graph (400X) showing HE staining results of small intestine morphology of mice in different treatment groups, wherein FIG. 3A is a blank control group; FIG. 3B: a sensitization model group; FIG. 3C: GB0.1 mg/d low dose group; FIG. 3D: GB0.8 mg/d high dose group; FIG. 3E: FAFH-2 control group;

FIG. 4 is a histogram of the serum IgE levels in mice of different treatment groups;

FIG. 5 is a bar graph of IgG and IgG subtype levels in mouse serum;

FIG. 6 is a graph showing the results of measurement of mouse spleen cell-released factor;

FIG. 7 is a graph showing the results of flow cytometry on the number of T cell subsets, wherein FIG. 7A is the proportion of Th1 in each group; FIG. 7B shows the proportion of Th2 in each group; FIG. 7C shows the proportion of Th17 in each group; fig. 7D is the proportion of fluorescent antibodies Treg for a representative mouse sample in each group; FIG. 7E shows the percentage of Th1, Th2 subtypes in each group; figure 7F is the percentage of Th17, Treg subtypes in each group;

FIG. 8 is a standard curve for protein concentration by BCA;

FIG. 9 is a graph showing the result of detecting mTOR-related protein in mouse splenocytes, wherein FIG. 9A shows the total protein and the corresponding phosphorylated protein bands in the mTOR signaling pathway; FIG. 9B is a bar graph showing the expression levels of mTOR, AKT, S6K1, SGK1 and 4E-BP1 as total proteins in mouse splenocytes; FIG. 9C is a bar graph showing the expression levels of the proteins p-mTOR, p-AKT, p-S6K1, p-SGK1 and p-4E-BP1 in mouse splenocytes.

Detailed Description

in the invention, the CAS number of ginkgolide B is 15291-77, and the molecular formula is C₂₀H₂₄O₁₀Molecular weight of 424.4,. The purity of the ginkgolide B is not particularly limited in the present invention, and the purity of ginkgolide B known in the art can be used. In the embodiment of the invention, the purity of the ginkgolide B is more than or equal to 98%, and the ginkgolide B is purchased from Solebao company.

In the invention, a food allergy mouse model is taken as an experimental object, and the ginkgolide B can effectively improve food allergy symptoms, reduce anaphylactic reaction in the mouse model and show similar treatment effect to FAHF-2. Therefore, the invention protects the application of the ginkgolide B in preventing and/or treating food allergy diseases. Meanwhile, the invention also defines the influence of the ginkgolide B on mTOR signal pathway protein in a food allergy mouse model, and the result shows that compared with a blank group of mice, the protein expressions of p-mTOR, p-AKT, p-SGK1, p-S6K1 and p-4E-BP1 of the mice in an allergic group are all obviously reduced, and meanwhile, the related protein expressions are increased by a GB administration experimental group; this indicates that ginkgolide B achieves the purpose of suppressing food allergy by interfering with mTOR signaling pathway.

In the present invention, in order to enrich the variety of food allergy drugs in the prior art, the use of the ginkgolide B preferably in combination with a food allergy inhibitor for the preparation of a drug for the prevention and/or treatment of food allergy. The food allergy inhibitor preferably comprises a traditional Chinese medicine compound food allergy inhibitor and a western medicine food allergy inhibitor. The traditional Chinese medicine compound food allergy inhibitor preferably comprises one or more of the following components: FAHF-1, FAHF-2, FAHF-3 and CRFC-1. The source of the traditional Chinese medicine compound food allergy inhibitor is not particularly limited, and the source of the traditional Chinese medicine compound food allergy inhibitor known in the field can be adopted. In the present example, the FAHF-2 was purchased from department of medicine, zunyi university of medicine. The western medicine food allergy agent preferably comprises at least one of the following components: adrenaline, levocetirizine hydrochloride, fexofenadine, diphenhydramine hydrochloride, external calamine lotion and the like. The kind of the food is not particularly limited in the present invention, and may be any kind of common food known in the art that is easily allergic, for example, the food preferably includes eggs, dairy products, dried fruits, seafood, beef and mutton, sesame, soybean and related products.

The present invention is not particularly limited in the kind and dosage form of the drug, and a drug dosage form well known in the art may be used. The method for preparing the drug is not particularly limited, and the method for preparing the drug well known in the field can be adopted. The content of the ginkgolide B in the medicine is not particularly limited, and when the medicine is taken later, the administration dose is not lower than 0.05mg/d (the administration object is a mouse), more preferably 0.1-0.8 mg/d, and most preferably 0.1 mg/d.

The invention provides a food allergy inhibitor, which takes ginkgolide B as a medicinal active ingredient. The active ingredients of the medicine preferably also comprise a traditional Chinese medicine compound food allergy inhibitor. The traditional Chinese medicine compound food allergy inhibitor preferably comprises one or more of the following components: FAHF-1, FAHF-2, FAHF-3 and CRFC-1.

In the invention, experiments prove that when the ginkgolide B is used as the only active pharmaceutical ingredient for treating the mouse model with food allergy, the results show that the ginkgolide B can effectively improve the symptoms of the food allergy and simultaneously can reduce the anaphylactic reaction in a mouse body, so that the ginkgolide B has the effect of inhibiting the food allergy.

The invention has no limit effect on the auxiliary materials, the preparation formulation and the preparation method of the food allergy inhibitor, so the auxiliary materials, the preparation formulation and the preparation method of the inhibitor which are well known in the field can be adopted. The food allergy inhibitor can effectively treat diseases caused by food allergy, and can be applied to preparation of medicaments for preventing and/or treating food allergy.

The following examples are provided to illustrate the use of bilobalide B of the present invention in the preparation of a medicament for preventing and/or treating food allergy and a food allergy inhibitor, but they should not be construed as limiting the scope of the present invention.

Example 1

Food-allergic mouse molding method

The egg white is used together with an adjuvant to successfully establish a mouse food allergy model, and the specific method comprises the following steps:

in the sensitization period, 5mg +10 mug of cholera toxin is perfused into egg white every week, and the sensitization induction time is as follows: on days 7, 14, 21 and 28, day 35, challenge with 20mg egg white solution and observation of sensitization symptoms in each group of mice was performed 30min after challenge. A blank control group was also set up and each mouse was gavaged with 300. mu.l PBS per time.

The mouse serum IgE levels were measured using an indirect ELISA detection kit (purchased from Sigma, USA).

The results are shown in FIG. 1. As can be seen from the results in fig. 1, the IgE level of the mice in the sensitized group is significantly increased (P is less than 0.05) compared with that in the blank control group, the ratios to the blank control group are both significantly greater than 2, and by combining the swelling symptoms around the mouth and nose of the mice observed in the mouse feeding process, the mice with food allergy successfully constructed in this embodiment are modeled, and the subsequent detection of relevant indexes can be completed by using this batch of mouse models.

Example 2

40 female BALB/c mice models with age of 6 weeks are selected as a sensitized group, and 10 female BALB/c mice with age of 6 weeks and normal growth are selected as a blank control group to be fed adaptively for one week. Mice in the sensitized group were divided again into a priming group (GB0.1 mg/d low dose group, GB0.8 mg/d high dose), a positive control group (FAHF-2:12mg/d) and a model group (0.9% physiological saline). It is expected that mice will be dosed once daily by gavage. The treatment is administered for 35 days.

In allergic mice, the body reacts differently after being stimulated by allergens, and the mice are characterized by dysphoria, scratching, mouth edema, respiratory distress and even shock death. Therefore, allergy symptom score is an important basis for characterizing allergic reactions. Mice in each treatment group were scored for allergic symptoms, using a scoring scale (table 1).

TABLE 1 mouse allergy symptom scoring sheet

The behavior reactions of the mice in the blank control group before and after the excitation are almost consistent, and corresponding allergic symptoms are not shown; in the mice of the model group, all the mice have the conditions of obvious edema of mouth, respiratory distress and reduced activity, wherein 2 mice have bluish purple mouths and 1 mouse has spasm; in mice of each group of GB interventions, the above allergic symptoms were improved, with only a small edema in the mouth of some mice, and a large number of others showing slight scratching and rubbing of the nose and head. From the results, it can be seen that the mouse allergic symptoms after GB drying are significantly improved, and the score of the mouse allergic symptoms is the same as that of the positive drug FAHF-2 group, indicating that GB has a good inhibitory effect on the symptoms of food-allergic mice (see fig. 2).

Example 3

Effect of GB intervention on mouse intestinal morphology

Mice from each treatment group of example 2 were discontinued 35 days after administration, challenged with egg white (to mimic the reentry of the allergen into the body to observe its response) at week 2 after discontinuation and sacrificed. The mouse small intestine is sliced, and the slices are stained by HE, and the shapes of the mouse small intestine slices of each treatment group are observed.

The results are shown in FIG. 3. Compared with a blank control group, the intestinal mucosa villus of the model group is obviously edematous and arranged irregularly, and the intestinal mucosa villus edema of the GB0.1 mg intervention group is obviously improved compared with the model group and is close to the effect of a positive control FAHA-2 group, so that the intestinal morphology of the GB0.1 mg intervention group is obviously improved and is better than that of the GB0.8 mg intervention group and is close to the effect of a positive drug FAHF-2.

Example 4

Analysis of specific antibody IgE levels in mouse sera

Mice in each treatment group of example 2 were discontinued 35d after administration, mice were challenged and sacrificed at 2 weeks after discontinuation, and sera were collected. Serum titer (IgE) levels were measured by ELISA (Sigma, USA).

Serum specific IgE levels for each group of mice are shown in FIG. 4. As can be seen, the IgE level of each intervention group is lower than that of the model group, and the IgE antibody level in the GB0.1 mg intervention group is obviously lower than that in the GB0.8 mg intervention group (P is less than 0.05), so that the similar effect is shown with the FAHF-2 intervention group. Therefore, the single traditional Chinese medicine GB0.1 mg/d has better inhibition effect on food allergy.

Example 5

Levels of specific antibodies IgG and IgG subtypes in mouse serum

Serum titer (IgG and IgG subtype) levels were measured by ELISA using each treatment group mouse serum collected in example 4 as a sample.

The results are shown in FIG. 5. The detection result of the specific antibody IgG and IgG subtype levels in the mouse serum shows that the IgG, IgG1 and IgG2a antibody levels in the serum of the GB0.1 mg intervention group are obviously reduced (P is less than 0.05) compared with the mice of the sensitization model group.

Example 6

Mouse splenic cytokine detection

Spleens of mice of each treatment group sacrificed in example 2 were collected, cultured spleen cells were isolated by a conventional method, and the level of splenocytes (IL-4, IL-10, IL-13, IL-17A, IFN-. gamma.) was measured using a kit (completed using a kit manufactured by Thermo Fisher Scientific Co., U.S.A.).

The results of the detection of the mouse spleen cytokines are shown in fig. 6, and compared with the blank group of mice, the detection of the corresponding spleen cytokines of the sensitized group of mice has statistical significance (P < 0.05). Compared with mice in a model group and a GB0.8 mg group, the levels of IL-4, IL-13, IL-17A and IFN-gamma of a traditional Chinese medicine GB0.1 mg intervention group are obviously reduced (P is less than 0.05); compared with the FAHF-2 group, the IFN-gamma and IL-4 levels are significantly different (P <0.05), while the IL-13 and IL-17A levels are the same; although there was a trend towards a decrease in IL-10 cytokine levels, the differences were not statistically significant. This indicates that GB treatment can significantly reduce splenocyte cytokine levels with an effect substantially comparable to that of FAHF-2.

Example 7

Mouse spleen cell flow cytometer for detecting T cell subgroup number

The operation steps are as follows: (1) preparation of mouse spleen single cell suspension: connective tissue was removed, washed repeatedly with PBS, spleen tissue was placed on a 200 mesh cell sieve with a 5ml plunger, gently rubbed and ground with a 5ml plunger, filtered, placed in a petri dish and washed with PBS, the cell suspension was centrifuged at 1500rpm for 5min, and the supernatant was discarded. Adding 3 times volume of RBC lysate, homogenizing, incubating at 4 deg.C for 30min, centrifuging at low temperature (1500rpm, 4 deg.C, 5min), adding 5 times volume of incomplete culture medium to stop reaction, centrifuging, removing supernatant, and resuspending with PBS to obtain 1ml single cell suspension; (2) cell treatment: dividing the cell suspension in each group of original tubes into two tubes (marked as No. 1 and No. 2), and adjusting the cell concentration to be 1 × 106/ml; (3) extracellular staining: adding CD4 antibody and CD4+ CD25 mixed antibody into No. 1 tube and No. 2 tube, and incubating for 30min at 4 ℃ in a dark place; (4) washing: adding 1ml PBS, mixing, centrifuging at 3000rpm for 5min, and removing supernatant; (5) cell fixation/rupture of membranes: adding 500 mul of membrane rupture/fixing solution into each tube, incubating for 30min at 4 ℃ in the dark, and washing; (6) intracellular staining: and (3) discarding the supernatant, adding a certain amount of IL-4, IL-17A and IFN-gamma mixed antibodies with different fluorescent labels into the No. 1 tube of each group, adding a certain amount of Foxp3 antibodies into the No. 2 tube of each group, and incubating for 30min at 4 ℃ in a dark place. (7) And (3) analysis: add 300. mu.l of flow-through supernatant per tube and detect in dark place) the number of mouse spleen T cell subsets (Th1, Th2, Th17, Treg).

The results are shown in FIG. 7. Compared with mice in a sensitized group, the traditional Chinese medicine GB0.1 mg in the intervention group has the advantages that the numbers of Th2 and Th17 are remarkably reduced (P <0.05), and the ratio of Foxp3+ Treg is remarkably increased (P < 0.05); compared with GB0.8 mg group, the number of Th17 is reduced remarkably, the number of Treg cells is increased remarkably (P < 0.05); the number of each T cell subset varied similarly to the FAHF-2 group.

Example 8

Influence of traditional Chinese medicine intervention on mTOR signal pathway protein of splenocytes of sensitized mice

The expression of mTOR and downstream proteins AKT, p-AKT, SGK1, p-SGK1, S6K1, p-S6K1, 4E-BP1 and p-4E-BP1 is detected by adopting a WesternBlot method, and the specific method is as follows:

1. protein extraction and concentration determination

The spleen cell culture plate cultured for 48h was taken out, the supernatant was discarded, the cells were gently washed 2 times along the well with precooled PBS, the residual PBS was carefully aspirated away with a small-range pipette, the lysate prepared in advance (RIPA: PMSF: phosphatase inhibitor protein: protease inhibitor ═ 100: 1: 1: 1) was added to sufficiently cover the cells, the cells were repeatedly aspirated on ice with the pipette to separate the cells from the plate wall, and sufficiently lysed on ice for 30 min. Transfer to a pre-cooled centrifuge tube. Then centrifuged in a low temperature centrifuge (12000 Xg, 4 ℃, 20 min). The supernatant was retained and the precipitate was discarded.

Determination of Total protein concentration by BCA method

(1) Preparing a standard substance: mu.L of the protein standard was taken and diluted to 120. mu.L with PBS buffer to give a concentration of 0.5 mg/mL. A96-well plate was taken and reagents were added according to Table 2, with 3 duplicate wells for each experimental group.

TABLE 2 preparation of protein standards at different concentrations

(2) Preparing a BCA working solution: according to standard holes and sample quantity, according to the liquid A: 50 of the liquid B: preparing working solution according to the proportion of 1, uniformly mixing, and keeping at room temperature for later use within 24 hours.

(3) Samples to be tested were diluted 10 and 20 times with PBS buffer solution in a gradient manner to a total volume of 20. mu.L, and then 200. mu.L of LBCA working solution was added to each well, and 3 duplicate wells were set for each experimental group.

(4) The 96-well plate was shaken manually for 30s and mixed well, and incubated in a 37 ℃ incubator for 30 min.

(5) And (3) measuring the OD value of each hole at 562nm of a full-automatic enzyme standard instrument by taking a No. 0 standard tube as a reference.

(6) A standard curve is drawn according to the protein content and the OD value, and the content (mu g) of each experimental histone is calculated as shown in figure 8.

(7) And calculating the actual concentration of the sample according to the formula I according to the dilution times of the sample.

Actual concentration of sample (μ g/μ L) × (20 μ g of each experimental histone/total volume of sample diluent) × sample dilution factor formula I

3. Electrophoretic sample loading preparation

Mixing the sample protein and 5 Xprotein sample buffer solution at a ratio of 4:1, boiling in metal bath at 100 deg.C for 10min for protein denaturation, and calculating the sample loading amount according to 30 μ g protein per well.

4. Glue making

TABLE 3 preparation of sodium dodecyl sulfate-Polyacrylamide gels

5. Glue pouring and sample loading

(1) Cleaning a glass plate: washing with tap water for several times, soaking and washing with pure water for one time, and air drying. (2) Pouring glue and loading sample: aligning two thick and thin glass plates, vertically clamping the two glass plates on a glue making frame, and respectively injecting distilled water before glue pouring for checking whether liquid leaks or not, and if not, respectively injecting distilled water; and (4) liquid leakage, namely shaking the prepared separation gel uniformly according to the recommended amount, filling the separation gel, and slowly paving a layer of double-distilled water liquid seal on the upper layer of the separation gel.

(3) After a significant fold line between the separation gel and water had formed (about 30min), the gelatin was said to have solidified, the upper layer of distilled water was decanted, and the excess double distilled water was blotted with filter paper.

(4) And (3) uniformly mixing the concentrated glue according to the recommended amount, injecting the mixed glue into the upper layer of the separation glue, and inserting a 10-hole comb (so as to avoid the generation of bubbles). And (5) slowly pulling out the comb vertically upwards after the comb is solidified.

(5) Carefully taking out the glass plate filled with the glue, clamping the glass plate in an electrophoresis clamp, then putting the glass plate into an electrophoresis tank, slowly pouring an electrophoresis buffer solution between two pieces of glue, checking whether the glue leaks or not, then filling the electrophoresis buffer solution, and preparing for sample loading.

(6) And (3) adding the sample to the sample hole by using a small-range pipettor, adding 5 mu l of rainbow Maker into the sample hole of the first lane, replacing the gun head of each group of samples once, and avoiding overflowing the sample hole during sample adding.

6. Electrophoresis

Firstly, setting the voltage to be 70V, regulating the voltage to be 120V after 30min and separating the red strip of the Maker, continuing electrophoresis for 70min, and stopping electrophoresis when the dye is indicated to be 3-5mm away from the bottom of the gel.

7. Protein transfer membrane

Preparing filter paper and a PVDF membrane with the same size as the gel, soaking the PVDF membrane in anhydrous methanol in advance for activation for 5min, and then placing an electric transfer clamp with the filter paper in precooled electric transfer liquid. The glass plate was removed from the bath and carefully pried open, the upper gel-concentrated portion was cut off, and the gel was carefully transferred from the glass plate to a pre-chilled electrotransfer for equilibration. According to the arrangement of wet type membrane conversion sandwiches, a membrane conversion sandwich is prepared from a negative electrode, sponge, filter paper, glue, PVDF membrane, filter paper and sponge to a positive electrode in sequence, an electric conversion device is installed, the sandwich is placed in an electric conversion tank, precooling electric conversion liquid is filled in the electric conversion tank, the electric conversion tank is placed in a foam box, an appropriate amount of ice bags are placed, the condition that the temperature is too high in the membrane conversion process is avoided, and the electric conversion is carried out for 180min under the condition of a constant current of 250 mA.

8. Membrane closure

After the end of the electrotransformation, the PVDF membrane was removed and placed in 6% skimmed milk (phosphorylated protein in 5% bovine serum albumin, since the skimmed milk contains casein), and the shaker was closed at low speed for 2h at room temperature. After blocking, washing with TBST three times was performed to perform antibody incubation experiments.

9. Primary antibody incubation

The primary antibody was diluted to 5mL using TBST according to the dilution factor recommended by the antibody specification and placed in a 10mL centrifuge tube, and the corresponding bands were cut and placed in the centrifuge tube containing the antibody as indicated by marker on the PVDF membrane, and left to stand overnight at 4 ℃.

10. Incubation with secondary antibody

(1) The membrane was removed from the primary antibody and placed in TBST and washed 3 times 10min each on a shaker.

(2) The secondary antibody was diluted to 10mL by the recommended fold with TBST and mixed well in a 50mL centrifuge tube, the TBST was blotted onto a dry film with filter paper, the strip was placed in the secondary antibody centrifuge tube and incubated for 2h at room temperature.

11. Color development and exposure

(1) Taking out the membrane after incubating the secondary antibody, washing the membrane for 3 times by TBST, 10min each time;

(2) under a light-shielding state, according to the volume ratio of the solution A to the solution B of 1: 1, preparing a luminous working solution;

(3) and taking the membrane out of the TBST, sucking dry the filter paper, placing the membrane in a special exposure box, uniformly covering the membrane with exposure liquid by using a pipettor, and automatically exposing the membrane in an all-purpose imaging system.

The results are shown in FIG. 9. Compared with mice in a blank group, the expression of mTOR and downstream total protein thereof has no significant difference in each experimental group, and the expression of P-mTOR, P-AKT, P-SGK1, P-S6K1 and P-4E-BP1 proteins in mice in a sensitized group is obviously reduced (P < 0.05). Compared with sensitized mice, the protein expression of P-mTOR and P-AKT of the traditional Chinese medicine GB0.1 mg intervention group is obviously increased (P <0.05), and the protein expression is similar to that of FAHF-2. Compared with GB0.8 mg intervention group, GB0.1 mg intervention group P-mTOR, P-SGK1 and P-4E-BP1 protein expression is higher (P <0.05), and P-S6K1 and P-AKT protein expression have no significant difference.

From the above examples, it can be seen that the compound GB can improve food allergy symptoms in mice, reduce allergic reactions in the mouse body, and inhibit food allergy, especially at a dose of 0.1mg/d, which shows similar therapeutic effects to FAHF-2. The experimental result shows that the intervention of GB on food allergy is related to the mTOR signaling pathway, and GB realizes the effect of inhibiting food allergy by improving the expression level of mTOR signaling pathway protein, so that GB can be combined with a traditional Chinese medicine compound food allergy inhibitor common in the prior art, and can also be combined with a reagent for improving the expression level of the mTOR signaling pathway protein to treat food allergy diseases.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. The application of ginkgolide B in preparing a medicament for preventing and/or treating food allergy is disclosed, wherein the structure of ginkgolide B is shown as formula I:

2. use according to claim 1, wherein the ginkgolide B is used in combination with a food allergy inhibitor for the preparation of a medicament for preventing and/or treating food allergy.

3. The use of claim 2, wherein the food allergy inhibitor comprises a traditional Chinese medicine compound food allergy inhibitor and a western medicine food allergy inhibitor.

4. The use of claim 3, wherein the herbal compound food allergy inhibitor comprises one or more of the following components: FAHF-1, FAHF-2, FAHF-3 and CRFC-1.

5. The use according to claim 3, wherein the western medicine food allergy inhibitor comprises at least one of the following components: epinephrine, levocetirizine hydrochloride, fexofenadine, diphenhydramine hydrochloride, and topical calamine lotion.

6. The use according to any one of claims 1 to 5, wherein the food comprises eggs, dairy products, dried fruits, seafood, beef and mutton, sesame, soy and related products.

7. A food allergy inhibitor is characterized in that bilobalide B is used as a pharmaceutically active ingredient.

8. The food allergy inhibitor according to claim 7, wherein the pharmaceutically active ingredient further comprises a herbal compound food allergy inhibitor.

9. The food allergy inhibitor according to claim 8, wherein the traditional Chinese medicine compound food allergy inhibitor comprises one or more of the following components: FAHF-1, FAHF-2, FAHF-3 and CRFC-1.