CN114903941B

CN114903941B - Dried orange peel alcohol extract and application thereof in treatment of eosinophilic esophagitis

Info

Publication number: CN114903941B
Application number: CN202210436040.5A
Authority: CN
Inventors: 曹明卓; 高勇; 王泽茜; 井贺云; 孟驿佳; 王媛
Original assignee: Henan University of Traditional Chinese Medicine HUTCM
Current assignee: Henan University of Traditional Chinese Medicine HUTCM
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2024-01-30
Anticipated expiration: 2042-04-24
Also published as: CN114903941A

Abstract

The invention discloses a tangerine peel alcohol extract and application thereof in treating eosinophilic esophagitis, and experiments prove that Th2 cytokine signal transduction is a necessary condition for inducing experimental EoE. The key effects of Th2 cytokines Interleukin (IL) -4, IL-5, IL-13 and TNF-alpha in EoE pathogenesis are proved, and the pathways of TGF-beta, MAPK and the like are involved in the occurrence of inflammation, and the tangerine peel alcohol extract prepared by the invention can effectively reduce the expression of proteins of TGF-beta, smad-3 and the like by inhibiting Th2 type inflammatory factors so as to achieve the aim of treating eosinophilic esophagitis.

Description

Dried orange peel alcohol extract and application thereof in treatment of eosinophilic esophagitis

Technical Field

The invention relates to preparation and application of a medicinal extract, in particular to a dried orange peel alcohol extract and application thereof in treating eosinophilic esophagitis.

Background

Eosinophilic esophagitis (EoE) is a chronic inflammatory esophageal disease associated with food antigen induction, which may be accompanied by other allergic diseases such as asthma, the pathophysiology of which involves allergen-driven Th2T cell immune responses. EoE was first identified in 1978 as an independent disease, whose major histological manifestation is infiltration of the esophageal mucosa layer by a large number of eosinophils (EOS for short), with esophageal dysfunction as the major clinical symptom. The disease can occur in all ages, adults and children under 50 years old are more common, especially children have higher incidence rate, and men are more than women (3-4:1). The clinical symptoms of adult patients are dysphagia, esophageal stenosis, food incarceration and reflux-like symptoms; children mainly show refusal to eat and malnutrition, seriously influence the life quality of patients and families thereof, and limit the social activities of partial patients to a certain extent. EoE distribution is unbalanced, and the prevalence of non-Spanish white people in developed countries such as North America, europe, and Australia is relatively high, and has increased to 1/1000 in these areas; the number of cases in asia (including the middle east) is currently relatively small. To date, no EoE epidemiological data exists in China, but as doctors have deeper and deeper knowledge of the disease, the research for clinically reporting the case also shows a remarkable increasing trend in China.

EoE induces a large number of factors, such as exposure to specific food allergens or airborne allergens, and its pathogenesis is generally thought to be consistent with other atopic diseases such as food allergy, asthma, allergic rhinitis, and atopic dermatitis, etc., and is triggered by Th2 immune responses. The inflammatory profile of EoE is characterized by an increased number of T cells and mast cells infiltrating the esophageal mucosa, and by high levels of expression of IL-5 and TNF- α, characterized by a Th2 immune response.

MAPK is an important transmitter of signal transmission from the cell surface to the inside of the cell nucleus, is a group of serine-threonine protein kinase which can be stimulated by different cells, not only regulates Th1/Th2 differentiation and eosinophil chemotactic factor expression, but also participates in immune response, and can accelerate the generation of inflammatory cytokines after activation, thereby exacerbating inflammatory response, wherein p38 mediates inflammation, apoptosis and the like, and is commonly used as a classical target for developing anti-inflammatory drugs. The increase of TGF-beta expression is a common way of fibrosis, and the biological function research of TGF-beta mainly has important regulation effects on the growth differentiation and the immune function of cells in the aspects of inflammation, tissue repair and the like in recent years. TGF- β1 belongs to one of the TGF- β superfamily members that regulate cell growth and differentiation, while the Smads protein family is an important intracellular signaling and regulatory factor that can cause fibrotic proliferation at many sites as a mediator of TGF- β1. The TGF-beta/Smad signaling pathway is the main classical pathway for TGF-beta to exert biological effects, and in addition, TGF-beta can activate the MAPK signaling pathway in a way other than the Smad signaling pathway. There are many gaps in the current understanding of the molecular mechanisms and immunopathology of EoE, but a number of clinical results suggest that EoE triggers eosinophil (Eos) infiltration into the esophagus, leading to inflammation, remodeling and fibrosis. The three subfamilies are likely to be involved in the pathogenesis of EoE, inhibiting the expression of p38 MAPK, TGF- β1 and smad3 proteins in this pathway, effectively reducing inflammatory factor release, and could be potential pathways for the treatment of EoE.

The pathogenesis of EoE has not been completely clarified so far, and is not completely cured at present, and the current treatment aims at improving clinical symptoms of patients, preventing complications (especially fibroproliferative stenosis of esophagus) and improving the life quality of patients. For its pathogenesis, eoE treatment methods are mainly dietetic and pharmaceutical treatments. Diet therapy can alleviate inflammation associated with EoE pathogenesis to some extent by avoiding allergens in the food, requiring major changes to the patient's daily life and eating habits, often with the problem of poor compliance. There is no specific drug approved by the FDA for treating EoE at present, and the first-line drug for treating EoE clinically is a local (inhaled)/oral (systemic) glucocorticoid drug because the pathogenesis of the specific drug is similar to that of allergic diseases such as asthma, atopic dermatitis and the like. Hormone therapy has been reported to alleviate oesophageal inflammation to some extent, but is prone to relapse after withdrawal, but studies have also shown that most patients receiving topical glucocorticoid therapy eventually fail to achieve complete normal oesophageal function, and if hormone therapy is received for a long period of time, exacerbation of eosinophilia in the oesophagus can instead occur; and patients (especially pediatric patients) have to be faced with chronic glucocorticoid therapy and unavoidable hormonal adverse effects (e.g., adolescent hypoevolutism, oropharyngeal/esophageal candidiasis, bone density abnormalities, glaucoma, hyperglycemia, and other antagonism, etc.).

Therefore, research on a novel medicine for treating eosinophilic esophagitis with definite and safe curative effect is a problem to be solved urgently.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides the dried orange peel alcohol extract and the preparation method thereof, which can achieve the aim of treating EoE by inhibiting Th2 inflammatory factors and reducing TGF-beta expression, and have good treatment effect in the application of treating eosinophilic esophagitis.

The purpose of the invention is realized in the following way:

an alcohol extract of pericarpium Citri Tangerinae, and its preparation method is as follows: weighing 50g of dried orange peel, shearing, placing in a 500ml round bottom flask, soaking in 70% ethanol for 2h, extracting with 10 times of solvent under normal pressure for three times respectively, wherein the extraction time is 2h, 1.5h and 1h in sequence, filtering with 8 layers of gauze, mixing the extractive solutions, concentrating under reduced pressure until no alcohol smell exists, vacuum freeze drying, and preserving at-20deg.C; the extraction is carried out for 5 times in parallel, the extraction yield range is 36.91+/-0.64%, the RSD is less than 5.00%, and the repeatability is good.

The tangerine peel alcohol extract is applied to the treatment of eosinophilic esophagitis.

Has the positive beneficial effects that: existing studies suggest that EoE is closely related to food and environmental allergens, and that, in addition to genetic and natural environments, th 2-type immune abnormalities (hyperactive) play a critical role in the development and progression of EoE and are closely related to IgE-mediated type i responses. Experiments prove that peanut allergen and skin irritation are necessary conditions for inducing experimental EoE. The key effects of Th2 cytokines interleukin-4 (IL-4), IL-5, IL-13 and TNF-alpha in the pathogenesis of EoE and the inflammatory signal paths of TGF-beta, MAPK and the like are also involved in the pathogenesis of EoE, and the tangerine peel alcohol extract prepared by the invention can effectively reduce the expression of proteins of TGF-beta 1, smad-3 and the like by inhibiting Th2 inflammatory factors and reducing the production of specific IgE, thereby achieving the aim of treating eosinophilic esophagitis.

(1) The invention provides a preparation method and an HPLC detection method of 70% dried orange peel alcohol extract, the content determination method has good repeatability, the pharmacodynamic substance basis for treating eosinophilic granulocyte esophagus is defined, and the main components of the method are hesperidin, nobiletin and hesperetin.

(2) The tangerine peel alcohol extract can be used for treating eosinophilic esophagitis, and the discovery overcomes the defect of lack of medicines for treating the eosinophilic esophagitis.

(3) In order to verify the effect of the dried orange peel alcohol extract on treating EoE, the invention provides an improved EOE mouse model, the mouse suffers from eosinophilic esophagitis by the method, the mouse model can basically meet the clinical symptoms and pathological manifestations of human EoE, the dried orange peel alcohol extract is adopted for treating the EoE, and the treatment result proves that the dried orange peel alcohol extract has a certain treatment effect on eosinophilic esophagitis and shows good safety.

Drawings

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

FIG. 2 is a thin-layer chromatography identification chart of the dried orange peel alcohol extract in the invention;

FIG. 3 is a high performance liquid chromatogram of the mixed standard and dried orange peel alcohol extract of the invention;

FIG. 4 is a graph showing the apparent indications of mice in the present invention;

FIG. 5 is a graph showing the change in body weight of mice in the present invention during the test period;

FIG. 6 is a graph showing the change in body temperature of each group of mice in the present invention within 40min after the last challenge;

FIG. 7 is a graph showing the change in allergy index of mice in each group after the last challenge according to the present invention;

FIG. 8 is a bar graph of spleen, lung, kidney index change at the end of treatment for each group of mice in the present invention;

FIG. 9 is a schematic of IL-4, IL5 and TNF- α levels (Th 2 cytokines) at the end of treatment in each group of mouse sera;

FIG. 10 is a schematic of IFN-gamma (Th 1 cytokine) and IL-10 (Treg cytokine) levels in the serum of each group of mice at the end of treatment;

FIG. 11 is a schematic of the serum of each group of mice in the present invention at the end of treatment for the levels of Penout-S-IgE;

FIG. 12 is a graph of H & E staining of esophageal, pulmonary and intestinal tissue at the end of treatment in groups of mice according to the invention;

FIG. 13 is a schematic representation of TGF- β1 levels in esophageal tissue at the end of treatment in groups of mice in the invention.

FIG. 14 is a schematic representation of TGF-beta 1, smad3 and p38 MAPK and their phosphorylated protein expression in various groups of mouse esophageal tissues.

Detailed Description

The invention is further described with reference to the drawings and detailed description which follow:

various studies have reported that allergic reactions can be produced by the airways, skin or gastrointestinal tract. The experiment is to establish a mouse EoE model through typical peanut protein food allergy, skin and gastrointestinal tract and other ways.

First aspect

1. Preparation of dried orange peel alcohol extract

Weighing 50g of dried orange peel, shearing, placing in a 500ml round bottom flask, soaking in 70% ethanol for 2h, extracting with 10 times of solvent under normal pressure for three times respectively, wherein the extraction time is 2h, 1.5h and 1h in sequence, filtering with 8 layers of gauze, mixing the extractive solutions, concentrating under reduced pressure until no alcohol smell exists, vacuum freeze drying, and preserving at-20deg.C. The extraction is carried out for 5 times in parallel, the extraction yield range is 36.91+/-0.64%, the RSD is less than 5.00%, and the repeatability is good.

1.1 thin layer chromatography identification

Test solution: adding methanol into pericarpium Citri Tangerinae ethanol extract powder to obtain 6mg/mL test solution, performing ultrasonic treatment for 20min, filtering with 0.22 μm microporous membrane, and collecting filtrate. Control solution: weighing appropriate amounts of hesperidin, nobiletin and hesperetin reference substances, and adding methanol to prepare corresponding saturated solutions respectively.

According to the thin-layer chromatography method under the item of dried orange peel decoction pieces in Chinese pharmacopoeia (general rule 0502), the four solutions are sucked, capillaries are respectively dotted on the same silica gel G plate, the two developing agents are respectively developed, finally, the two developing agents are taken out and dried, the aluminum trichloride test solution is uniformly sprayed, and the solution is subjected to inspection and photographing under an ultraviolet lamp.

1.2 conditions for high Performance liquid chromatography

1.2.1 chromatographic conditions and System applicability test

Octadecylsilane chemically bonded silica is used as a filler. Acetonitrile as mobile phase A and water as mobile phase B, gradient elution and full-wave detection were performed under the conditions shown in the following table.

TABLE 1.3 chromatographic conditions

(2) Preparation of control solution and test sample solution

Taking appropriate amount of hesperidin reference substance, nobiletin reference substance and hesperetin reference substance, precisely weighing, and adding methanol to obtain mixed solution containing hesperidin 0.4mg, nobiletin 25 μg and hesperetin 15 μg per 1 ml.

Taking coarse powder (passing through a second sieve) of about 0.2g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of methanol, sealing, weighing, performing ultrasonic treatment (power 300W; frequency 40 kHz) for 45 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, and collecting subsequent filtrate.

(3) Assay

Precisely sucking 5 μl of each of the control solution and the sample solution, loading, and measuring.

Second aspect

1 laboratory animal

SPF-class BALB/C mice 60, 7-8 weeks old, female, purchased from Jinan Pengyue laboratory animal Breeding Co., ltd., animal license number SCXK (Lu) 2019003, and laboratory unit used license number SYXK (Yu) 200-0004. Raising in laboratory animal house of applicant.

2 drugs, reagents and instruments

2.1 pharmaceutical products and reagents

2.2 instruments

Preparing a crude peanut protein extract: drying fresh peanut, peeling, pulverizing to powder with pulverizer, soaking in 100mL of precooled acetone in beaker, and defatting overnight at 4deg.C until supernatant is clear. And (3) after the acetone is air-dried, adding PBS buffer solution to extract protein, soaking for 24 hours in a refrigerator at 4 ℃, centrifuging for 20 minutes at 1500r & min & lt-1 & gt, removing residues on the lower fat layer, leaving a middle layer, and repeatedly centrifuging to obtain supernatant, namely the crude extract (PPE) of peanut protein. The concentration of PPE was determined using BCA protein quantification kit, and the specific procedure was performed according to the kit instructions. Freeze-drying and storing at-20deg.C.

PPE sensitization solution: 24mg of PPE is weighed according to the quantitative result of the protein, 240mg of aluminum adjuvant (40 mg/mL of stock solution) is weighed and dissolved into 12mL of PBS, and the existing preparation is fully dissolved.

PPE ointment mix: the PPE and calcipotriol ointment are uniformly mixed according to the weight ratio of 1:100, namely 0.2mg of PPE is contained in each 20mg of ointment mixture.

PPE excitation liquid: according to the quantitative result of protein, 300mg of PPE is weighed and dissolved in 12mL of PBS, and the on-the-fly preparation is fully dissolved. The last high dose of challenge solution was 600mg PPE in 12mL PBS.

Dried orange peel alcohol extract: ETP prepared by the method of 3 items was dissolved so that its concentration was 800mg/kg, 400mg/kg, 200mg/kg, respectively.

Dexamethasone solution: according to the drug instructions, the initial dose of the adult is 0.75-3.00 mg once, 2-4 times a day, and the maintenance amount is about 0.75mg (1 tablet) a day. According to the body surface area folding algorithm, the equivalent dose ratio of the mice to the human is 9.1, 1mg/kg is given in the early period of administration (the previous week), and 0.2mg/kg is continuously given in the later period of administration.

3 method

3.1 grouping, model preparation and administration

As shown in figure 1, in the experiment, BALB/c female mice are selected as experimental animals, PPE is adopted as allergen, the sensitization mode adopts combination of intraperitoneal injection and percutaneous injury, and the excitation mode selects esophageal perfusion excitation. The molding method refers to the method of the literature and is modified to build a model, as shown in fig. 1.

(1) Basic sensitization phase: immunization was established at weeks 0, 1, 2, and 3, respectively, and mice in the remaining groups except the blank group were intraperitoneally (i.p.) injected with 0.2mL of 2mg/mL of sensitization solution 1 time per week for 4 times; mice from the blank group were given equal amounts of PBS buffer at the same time.

(2) Transdermal sensitization phase: in addition to the blank, 20mg of PPE ointment mixture was applied for 1-7, 21-28 days, respectively, one week at a time; mice in the blank group were given an equal amount of calcipotriol ointment.

(3) Local stimulation phase: after the 4 th intraperitoneal injection, a local stimulation phase was performed on day 28 at 7 intervals. Each mouse of the remaining groups, except the blank group, was stimulated for esophageal perfusion, given 0.2mL25mg/mL of PPE challenge solution 1 time per week, 5 times total; mice from the blank group were given equal amounts of PBS buffer at the same time.

(4) And (5) modeling evaluation: the basic sensitization stage is carried out for 4 times, in order to preliminarily grasp whether modeling is successful or not, specific PPE-SIgE in the serum of the mice is detected by taking blood from the mandibular blood in the third week, and the mice are divided into 6 groups according to the detection result and into blank groups, model groups, dexamethasone groups and high, medium and low dose groups of the dried orange peel alcohol extract according to a random digital table method. Large doses of 25mg/mL challenge solution were given at 0.2mL each before sacrifice on day 57, and the end of each index experiment was recorded by observation.

Normal group (Naive): PBS buffer; model set (Sham): PBS buffer; dexamethasone group (Dex): dexamethasone solution; drug administration group: high dose (ETP-H concentration of 800 mg/kg), medium dose (ETP-M concentration of 400 mg/kg) and low dose (ETP-L concentration of 200 mg/kg) of the dried orange peel alcoholic extract.

3.2 changes in body weight and apparent signs

After the last challenge (day 56), the mice were blindly observed and recorded for their behavioral changes, i.e. allergy index (asymptomatic 0 min; harassment head and nose 1 min, diarrhea, periocular and mouth edema, hair uprighted, activity frequency reduced by 2 min, cyanosis, dyspnea 3 min, no or only mild response, tremors and cramps 4 min after stimulation, 5min of death).

And an infrared thermometer was used to record the body temperature change within 40min after the mice were challenged.

3.3 cytokine detection

After the apparent observation was completed, the mice were removed, blood was collected through the orbital venous plexus, and the serum was separated by centrifugation at 12000rpm/min for 10 min. The levels of PPE-s-IgE (peanut protein specific IgE), IL-4, IL-5, TNF- α, IFN- γ, IL-10 in the serum of mice were tested according to ELISA kit protocol.

3.4HE staining for the observation of lung histopathological changes

Mice were sacrificed, esophageal, pulmonary, and intestinal tissues were taken and fixed in 4% paraformaldehyde, paraffin embedded and sectioned for specimens, and hematoxylin-eosin (HE) staining of pathological specimens was performed. And observing inflammatory penetration, tissue damage, intestinal epithelial cell villus deformation, intestinal crypt loss pathological changes and the like in each tissue.

3.5Western blot detection of esophageal tissue TGF-beta 1, smad3 and p38 MAPK protein levels

After dissolution of the esophageal tissue of the mice in the sample buffer, the protein concentration was determined by BCA protein kit and an equivalent amount of protein sample was separated by SDS-PAGE. After separation the proteins were transferred to PVDF membranes. Then blocked with 5% nonfat dry milk solution and incubated overnight with antibody at 4 ℃. After washing, the membrane was incubated with horseradish peroxidase-labeled goat anti-rabbit II antibody for 2h at room temperature and protein bands were detected with ECL chemiluminescent detection reagent.

3.6 immunohistochemistry

Paraffin sections were analyzed for immunohistochemistry. I.e. after dewaxing fixation, TGF-beta 1 antibody and II antibody are added for incubation, and then the sealing piece is stained.

4 statistical treatment

Statistical analysis was performed using SPSS19.0 software. All data are expressed as mean ± standard deviation (mean ± SD). The comparison between the groups adopts one-way ANOVA, and the comparison between the groups adopts t-test. The difference of P <0.05 is statistically significant.

Results

1 dried orange peel alcohol extract quality control

The identification by thin layer chromatography shows the development results as shown in figure 2, (1 hesperetin reference, 2 Sichuan hesperetin reference, 3 hesperidin reference, 4 hesperetin alcohol extract) in figure 2a is a view of ultraviolet lamp at 365nm, and figure 2b is a view of ultraviolet lamp at 354nm, and in order to make spots more clear and visible, the images are ashed, and fluorescent spots with the same color appear in the sample chromatogram at the positions corresponding to those of the reference chromatogram.

As shown in FIG. 3, 70% ethanol extract of pericarpium Citri Tangerinae (1 hesperidin, 2 nobiletin, 3 hesperetin) is prepared by mixing control solution and test solution under HPLC condition, respectively injecting 5 μl, and recording chromatographic peak retention time and peak area within 25min, wherein hesperidin, nobiletin, and hesperetin are known as main components.

2 appearance index

Allergic reactions are systemic, even life threatening, diseases triggered by mediators released by mast cells and basophils, which are activated by allergic (IgE-mediated) or non-allergic (non-IgE-mediated) mechanisms. This is a rapidly evolving multi-system process involving the skin, lung, gastrointestinal and cardiovascular systems. In clinic, food allergy is often mediated by IgE, whereas EoE is mostly associated with food allergy.

The PPE-induced EoE mouse model has typical characteristics of food allergy and can well simulate clinical typical symptoms and pathological changes of eosinophilic esophagitis. Compared with the mice in the negative control group, the mice in the model group start to act slowly, have poor hair luster and loose stool after the second excitation, wherein some mice show obvious symptoms of bows, prostration, hair erection, lip swelling and the like (shown in figure 4).

The mice in the model group and the treatment group have allergic manifestations such as nasal scratching, diarrhea, cyanosis and the like with different degrees after each excitation; the negative control mice showed no allergic behavior except for occasional nasal scratching. After the last challenge, the allergic behavioural manifestations of the mice present (and scored according to a semi-quantitative score to score for the most severe behaviours present) were recorded for 15min, with specific scoring results shown in fig. 4. Compared with a negative control group, the allergic symptom score of the mice in the model group is obviously increased, the average score is divided into 2 scores, and the mice have extremely significant difference (p < 0.01); after the dried orange peel alcohol extract is treated, allergic symptoms are relieved to a certain extent, and the average scores of high, medium and low dose groups are 0.71,1 and 1.33 respectively, so that the dose dependency is shown. The high dose group mice had significantly lower allergy scores than the model group (sham) and had statistical differences (p < 0.01) comparable to dexamethasone, as shown in figure 4.

Figure 5 shows the weight change of the various mice throughout the experiment, with the average weight of the mice in the model group decreasing by 2.71g, starting from the weight decrease after the first challenge (28 days), to a different extent after each challenge. After PPE challenge, the model mice continued to decrease in body weight and there was no relief as the challenge progressed, the last model mice significantly decreased by 5.72g (P < 0.01) compared to the negative control.

The body weight of each tangerine peel alcohol extract treatment group is reduced to different degrees after the excitation, but the body weight reduction trend is obviously restrained, the dose dependence is shown, and the body weight reduction trend of the high-dose group is obviously restrained. The body weight was still slightly increased at the first 3 shots, and after the 4 th and 5 th (last) shots, the body weight was reduced by only 0.85 and 0.80g from the initial body weight at the end of the test. The medium dose group and the low dose group also show obvious weight loss in the last two shots. By the treatment endpoint, dexamethasone (P < 0.01), high and medium dose (P < 0.05) dried orange peel alcohol extract mice had a significant difference in weight loss from model mice, whereas high and medium dose dried orange peel alcohol extract mice were not statistically different from dexamethasone as shown in fig. 5.

As shown in fig. 6 a, each group of mice, except the negative control group, had a different degree of body temperature drop within 40min after challenge, and began to rise in body temperature after 10-30 min. As shown in fig. 6B, the model group mice had most significant body temperature reduction, which was 2.3 ℃ lower than the basal body temperature at 30 min; after the treatment of the dried orange peel alcohol extract, the degree of the obvious body temperature reduction is relieved to a certain extent, the temperature is reduced by only 0.4 ℃ in the high-dose group for 30min, and obvious body temperature rise occurs in 40min, and the temperature is reduced by only 0.07 ℃ compared with the basic body temperature.

Since eosinophilic esophagitis is an autoimmune disease, in the experimental process, the model group mice have the phenomena of lung inflammation and spleen enlargement, and the lung coefficient and the spleen coefficient of the model group mice are both obviously increased (p < 0.001); the lung coefficient and the spleen coefficient of the tangerine peel alcohol extract treatment group are also increased compared with those of mice in a negative control group, but compared with mice in a model group, the spleen coefficient and the lung coefficient of the tangerine peel alcohol extract treatment group are reduced to different degrees (p is less than 0.05), the tangerine peel alcohol extract can reduce the inflammation of the spleen and the lung to a certain degree, and can improve the in vivo immune balance to a certain degree.

The model group kidney index increased to some extent compared with the negative control group, but there was no statistical difference among the groups, indicating that the EoE animal model or the trend of making the kidney swelling, the dried orange peel alcohol extract can slightly alleviate the major trend of the kidney, as shown in fig. 8.

5.3 Biochemical index

The pathogenesis of EoE has not been completely clarified so far, and most researchers believe that EoE is a disease based on Th2 type immune response, and Th2 cytokine products IL-4, IL-5 and eosinophil effector all play key roles in EoE pathogenesis. Thus we examined the levels of cytokines such as IL-4, IL-5, TNF-. Alpha.L, IFN-y in the serum of each group of mice.

As shown in fig. 9 below, the concentrations of TNF- α, IL-4 and IL-5 cytokines were all significantly elevated in the serum of mice in the model group (p < 0.01) compared to mice in the negative control group; the corticoid dexamethasone can obviously reduce the level of each inflammatory cytokine, and the levels of IL-4, IL-5 and TNF-alpha cytokines are respectively reduced by 66.4%,51.2% and 77.7%. The dried orange peel alcoholic extract group treated each of the above inflammatory factors was reduced to a different extent compared to the model group, and had a dose dependency, and the high dose group reduced the IL-4, IL-5 and TNF- α cytokine levels by 61.0%,55.6% and 61.5%, respectively, on average, comparable to the dexamethasone group reduced levels, as shown in FIG. 9.

As shown in fig. 10 below, both IFN- γ and IL-10 levels in the serum of mice in the model group were significantly reduced (p < 0.01) compared to mice in the negative control group; dexamethasone increased IFN-gamma and IL-10 cytokine levels by 28.5% and 42.0%, respectively. The IFN-gamma and IL-10 anti-inflammatory cytokine levels are also significantly increased and dose dependent following treatment with the dried orange peel alcoholic extract group. The high, medium and low doses of the dried orange peel alcohol extract respectively raise IFN-gamma levels by 26.8%,31.9% and 32.5%, and IL-10 cytokine levels by 36.6%,17.7% and 13.8%.

IgE is a key immunoglobulin in the pathogenesis of IgE-mediated related allergic diseases, and although there is no data demonstrating the specific role IgE plays in the pathogenesis of EoE, igE levels are closely related to the prognosis and severity of eosinophils in EoE patients. Peanut-Specific IgE (Peanot-Specific-IgE, peanot-s-IgE) levels were measured by ELISA, as shown in FIG. 11 below. Compared with the negative control group, the serum Penout-s-IgE of the model group is obviously increased, the average concentration reaches 369.89ng/mL, and the model group has extremely obvious statistical difference (p < 0.0001). The Penout-s-IgE of the dried orange peel alcohol extract is reduced to different degrees, and the IgE level of the high-low dosage group is respectively reduced by 53.5%,43.5% and 26.1% and has dose dependency.

5.4 pathological section

Pathological changes of the EoE model mice can be intuitively evaluated by pathological sections. FIG. 12 shows H & E pathology in esophageal (A), pulmonary (B) and small intestinal (C) tissues at the end of treatment in each group of mice. Dexamethasone group was 0.5mg/kg dexamethasone group; the high, medium and low dosages are 800mg/kg, 400mg/kg and 200mg/kg of pericarpium Citri Tangerinae extract respectively.

H & E stained sections of esophageal tissue (a in fig. 12) showed: the negative control group mice have clear esophageal structure, even esophageal wall, slight inflammatory cell infiltration at the periphery, and no congestion in the esophagus. Model group mice have abnormal esophageal tissue structure, basal cell proliferation, connective tissue proliferation, and occasional hyperemia; the papilla and the spindle process of the lamina propria are prolonged, and the upper lamina propria is fibrosed and has a tendency to fall off; eosinophils are moist in a small amount, and the cell nuclei are obviously foliated, so that the method basically accords with the clinical pathological changes of EoE; the proliferation condition and fibrosis of dexamethasone and dried orange peel alcohol extract high-dose groups are obviously reduced, the lobed cell nucleus is reduced, the hyperemia phenomenon is avoided, and the low-dose groups are improved to a certain extent but have no obvious difference.

The H & E stained section results of lung tissue (B in fig. 12) show: the lung tissue airway structure of the mice in the negative control group is clear, the alveoli are complete, the alveoli are fine and uniform in interval, the alveoli wall is hyperemia, a small amount of inflammatory cells infiltrate around the trachea, and no epithelial cells drop in the trachea. The model group mice have abnormal lung tissue airway structure, basically disappeared alveolar structure, obviously thickened alveolar space, severe alveolar wall congestion, narrow airway lumen, scattered bronchial epithelial cells and exuded lymphocytes, and a large amount of inflammatory cell infiltration around the trachea; after the tangerine peel alcohol extract treatment, the lung tissue pathology is improved.

H & E stained section results of small intestine tissue (C in fig. 12) show: the small intestine tissue structure of the mice in the negative control group is clear, and submucosa is infiltrated by a little inflammatory cells occasionally, and the mice have no loose structure. The abnormal intestinal tissue structure of the mice in the model group is expressed by uneven villus of intestinal mucosa and different lengths; with significant, massive eosinophil infiltration; presents a certain degree of intestinal wall thickening, goblet cell increase, etc. After the treatment of the dried orange peel alcohol extract, intestinal villi of the small intestinal tissue of the high-dose group is recovered to be normal, no obvious inflammatory cell infiltration exists, the low-dose group in the dried orange peel alcohol extract is improved, and the phenomena of obvious damage to intestinal mucosa, falling of epithelial cells and the like still exist.

As shown in fig. 12D, TGF- β1 has the function of modulating immune responses and fibrosis, the most critical cytokine for mediating tissue inflammation, regulating extracellular matrix deposition. TGF- β1 cells stained localized to the cytoplasm and appeared yellowish-brown, diffusely distributed as positive expression of TGF- β1. The TGF-beta 1 has higher positive expression level in esophageal tissue of a model group mouse, the positive expression of a blank group mouse is lower, and the positive expression quantity of TGF-beta 1 of a dexamethasone group and a tangerine peel alcohol extract group is obviously reduced after treatment.

5.5 alcohol extract of dried orange peel on TGF-beta 1, smad3 and p38 MAPK protein levels in esophageal tissue of EoE model mouse model

Protein was extracted from each group of esophageal tissue, and after quantification, a western blot experiment was performed, and the results are shown in fig. 14. Compared with the blank group, the expression levels of TGF-beta 1, P-Smad3 and P-P38 MAPK proteins in esophageal tissues of the mice in the model group are all obviously increased, and the statistical difference exists (P < 0.05). Compared with the model group, the expression levels of TGF-beta 1, P-Smad3 and P-P38 MAPK proteins in the tangerine peel alcohol extract high and medium dose groups are reduced to different degrees (P < 0.05).

As shown in fig. 13, eoE is currently believed to be involved in the occurrence and development of the disease, such as food and environmental allergens, genetic and immune abnormalities (hyperactive), etc., and it is believed that EoE is mediated by IgE-mediated type i allergy and/or non-IgE, and it is experimentally confirmed that Th2 cytokine signaling is a necessary condition for inducing experimental EoE. The critical role of Th2 cytokines Interleukin (IL) -4, IL-5, IL-13 and TNF-alpha in EoE pathogenesis has been demonstrated, as well as the involvement of other cytokines such as TGF-beta, MAPK, etc.

Existing studies suggest that EoE is closely related to food and environmental allergens, and that, in addition to genetic and natural environments, th 2-type immune abnormalities (hyperactive) play a critical role in the development and progression of EoE and are closely related to IgE-mediated type i responses. Experiments prove that peanut allergen and skin irritation are necessary conditions for inducing experimental EoE. The key effects of Th2 cytokines interleukin-4 (IL-4), IL-5, IL-13 and TNF-alpha in the pathogenesis of EoE and the inflammatory signal paths of TGF-beta, MAPK and the like are also involved in the pathogenesis of EoE, and the tangerine peel alcohol extract prepared by the invention can effectively reduce the expression of proteins of TGF-beta 1, smad-3 and the like by inhibiting Th2 inflammatory factors and reducing the production of specific IgE, thereby achieving the aim of treating eosinophilic esophagitis.

The specific treatment results are as follows:

after the second excitation, the EoE model mice start to show obvious systemic allergic symptoms such as harassment of nose and head, hair erection, bow back and the like, and the allergic index score is obviously increased (P < 0.01); the levels of peanut protein specific IgE, igG1 and typical Th2 cytokines IL-4, IL-5, TNF-alpha, etc. in serum are obviously increased (P < 0.01), the levels of Th1 cytokines IFN-gamma and anti-inflammatory cytokines IL-10 are obviously reduced (P < 0.01), and compared with the mice in a negative control group, the mice in a model group have typical Th1/Th2 imbalance; obvious damage to esophagus, lung and intestine tissue, which is manifested by connective tissue hyperplasia, fibrosis, eosinophil infiltration, etc.; elevated P-P38 MAPK, TGF-. Beta.1 and P-smad3 protein expression in esophageal tissue (P < 0.05), and TGF-. Beta.1 positive expression (P < 0.01).

Compared with the mice in the model group, the dried orange peel alcohol extract can effectively relieve the conditions of weight loss, body temperature reduction, anaphylactic index rise and the like of the mice during the excitation period after the treatment; the levels of PPE-S-IgE, igG1, IL-4, IL-5, TNF-alpha and the like in the serum of the mice are reduced, the levels of IL-10 and IFN-gamma in the serum of the mice are increased, and the immune imbalance condition of Th1/Th2 is effectively regulated; the pathological damage to the tissues of esophagus, lung and intestine is relieved to a certain extent; the tangerine peel alcohol extract can inhibit activation of p38 MAPK and TGF-beta channels in esophageal tissue of a PPE model mouse, so that the expression of phospholyated-p 38 MAPK, phosphorylated-TGF-beta 1 and phospholyated-smad 3 is reduced, and the curative effect of the high-dose tangerine peel alcohol extract is equivalent to that of dexamethasone. Has certain intervention effect on eosinophilic esophagitis induced by peanut protein.

(1) The invention provides a preparation method and an HPLC detection method of 70% dried orange peel alcohol extract, the content determination method has high repeatability, the pharmacodynamic substance basis for treating eosinophilic granulocyte esophagus is defined, and the main components of the method are hesperidin, nobiletin and hesperetin.

(3) In order to verify the effect of the dried orange peel alcohol extract on treating EoE, the invention provides an improved EOE mouse model, the mouse suffers from eosinophilic esophagitis by the method, the mouse model can basically meet the symptoms and pathological manifestations of human EoE, the dried orange peel alcohol extract is adopted to treat the EoE, and the treatment result proves that the dried orange peel alcohol extract has a certain treatment effect on eosinophilic esophagitis and shows good safety.

Claims

1. The application of the dried orange peel alcohol extract is characterized in that: application of pericarpium Citri Tangerinae ethanol extract in preparing medicine for treating eosinophilic esophagitis is;

the preparation method of the dried orange peel alcohol extract comprises the following steps: weighing 50g of dried orange peel, shearing, placing in a 500ml round bottom flask, soaking in 70% ethanol for 2h, extracting with 10 times of solvent under normal pressure for three times, filtering with 8 layers of gauze for 2h, 1.5h and 1h, mixing the extractive solutions, concentrating under reduced pressure until no alcohol smell exists, vacuum freeze drying, preserving at-20deg.C, extracting for 5 times in parallel, wherein the yield range of the extract is 36.91+ -0.64%, RSD is <5.00%, and repeatability is good.