Use of formula food composition for special medical use suitable for infants allergic to food proteins for non-therapeutic purposes of ameliorating eosinophilic esophagitis

Abstract

The invention relates to the technical field of compositions, and particularly discloses an application of a special medical-purpose formula food composition suitable for infants allergic to food protein to the non-treatment purpose of improving eosinophilic esophagitis. The special medical use formula food composition suitable for infants allergic to food proteins comprises docosahexaenoic acid, eicosatetraenoic acid and nucleotides. The special medical formula food composition suitable for infants allergic to food protein provided by the invention can improve esophageal epithelial hyperplasia and reduce the level of IL-13 cell factors by increasing the number of bifidobacteria and reducing the number of enterobacter and eosinophilic granulocytes in esophageal tissue, so that the eosinophilic granulocytic esophagitis is remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of compositions, in particular to the use of a special medical purpose formula composition suitable for food protein allergic infants for non-therapeutic purposes to improve eosinophilic esophagitis. BACKGROUND

[0002] Eosinophilic esophagitis is a chronic, immune / antigen-mediated inflammation of the esophagus characterized by eosinophilic (EOS) infiltration of the esophageal mucosa layer (usually > 15 per high power field), which manifests as dysphagia, food impaction, reflux-like symptoms, etc. The occurrence of EoE is closely related to abnormal Th2 immune response of the body to specific food antigens.

[0003] Currently, in addition to drug treatment, the clinical nutrition scheme for eosinophilic esophagitis usually adopts a diet elimination therapy, such as a six-food elimination diet, which includes eliminating dairy products, wheat, eggs, soybeans, nuts, and seafood. Although this method is effective, it is extremely difficult to implement and severely affects the quality of life of patients, and repeated endoscopic evaluation is required to determine the allergenic food, which is a cumbersome and invasive process. Therefore, there is an urgent need for a new nutritional intervention scheme for eosinophilic esophagitis that is effective, safe, easy to implement, and can improve the quality of life of patients. SUMMARY

[0004] In view of this, the present application provides the use of a special medical purpose formula composition suitable for food protein allergic infants for non-therapeutic purposes to improve eosinophilic esophagitis. The composition provided by the present application can non-therapeutically regulate the immune pathways related to eosinophilic esophagitis, repair the esophageal mucosal barrier, and improve eosinophilic esophagitis, which has important clinical significance and application prospects.

[0005] To solve the above technical problems, the technical solution provided by the present application is: The present application provides the use of a special medical purpose formula composition suitable for food protein allergic infants for non-therapeutic purposes to improve eosinophilic esophagitis. The special medical purpose formula composition suitable for food protein allergic infants includes the following components: docosahexaenoic acid, eicosapentaenoic acid, and nucleotides.

[0006] The food protein allergic infants include infants who develop eosinophilic esophagitis due to food protein allergy.

[0007] The present application provides a special medical purpose formula composition for food protein allergic infants, which can produce systemic anti-inflammatory mediators, inhibit Th2 immune response in the whole body and esophagus, and further inhibit the recruitment and activation of eosinophils, thereby assisting in improving eosinophilic esophagitis; the inventors have further found that, when nucleotides are further added to the composition, the nucleotides can promote intestinal epithelial repair, reduce antigen leakage, help promote the differentiation of regulatory T cells, inhibit overactive Th2 immune response, reinforce the intestinal barrier, reduce food allergens into the blood, and further improve eosinophilic esophagitis. In addition, studies have shown that, through synergistic action, DHA, ETA and nucleotides in the composition can increase the abundance of beneficial bacteria, reduce the abundance of intestinal bacilli, reduce the infiltration and activation of eosinophils in the esophageal tissue, effectively reverse esophageal epithelial hyperplasia, and reduce the levels of IL-13 and other cytokines, thereby effectively relieving eosinophilic esophagitis.

[0008] Preferably, the mass ratio of DHA, ETA and nucleotides is (0.07-0.2):(0.07-0.4):(0.012-0.058).

[0009] The inventors have found that, when the components in the composition are controlled to a specific ratio, eosinophilic esophagitis can be further improved.

[0010] Further preferably, the mass ratio of DHA, ETA and nucleotides is (0.08-0.15):(0.08-0.35):(0.02-0.05).

[0011] Further preferably, the mass ratio of DHA, ETA and nucleotides is (0.09-0.1):(0.09-0.2):(0.03-0.04).

[0012] Further preferably, the mass ratio of DHA, ETA and nucleotides is 0.098:0.107:0.035.

[0013] Through a large number of experimental verifications, when the mass ratio of DHA, ETA and nucleotides in the composition is 0.098:0.107:0.035, the number of bifidobacterium can be significantly increased, the number of enterobacterium and esophageal tissue eosinophils can be reduced, esophageal epithelial hyperplasia can be improved for non-treatment purposes, the level of IL-13 cytokine can be reduced, and thus eosinophilic esophagitis can be significantly improved.

[0014] The nucleotide comprises the following mass percentage of each component: 30-40% of cytidine acid disodium, 20-28% of uridine acid disodium, 18-23% of adenosine monophosphate, 10-16% of inosine acid disodium, and 5-10% of guanosine acid disodium.

[0015] The present application defines the specific composition of the nucleotide, and by selecting the specific nucleotide, the eosinophilic esophagitis can be further improved.

[0016] Illustratively, the nucleotide is purchased from Nanjing Tongkai Shuoye Biotechnology Co., Ltd.

[0017] Illustratively, the docosahexaenoic acid and eicosapentaenoic acid are purchased from Runke Bioengineering (Fujian) Co., Ltd.

[0018] Preferably, the preparation method of the special medical purpose formula composition suitable for food protein allergic infants comprises the following steps: The weighed docosahexaenoic acid, eicosapentaenoic acid and nucleotide are uniformly mixed to obtain the special medical purpose formula composition suitable for food protein allergic infants.

[0019] The composition provided by the present application has a simple preparation method and can be prepared by simple mixing.

[0020] Preferably, the special medical purpose formula composition suitable for food protein allergic infants is a milk protein deep hydrolysis formula or an amino acid formula food.

[0021] The special medical purpose formula composition suitable for food protein allergic infants provided by the present application optimizes the composition and the ratio of the composition, improves the number of bifidobacterium, reduces the number of enterobacterium and esophageal tissue eosinophils, improves esophageal epithelial hyperplasia for non-therapeutic purposes, reduces the IL-13 cytokine level, and further significantly improves eosinophilic esophagitis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The figure is a statistical graph of the proportion of enterobacterium sequences in the intestinal flora of each group of mice; Figure 2 The figure is a statistical graph of the proportion of bifidobacterium sequences in the intestinal flora of each group of mice; Figure 3 The figure is a statistical graph of the number of esophageal tissue eosinophils of each group of mice; Figure 4 The figure is a statistical graph of the esophageal epithelial thickness of each group of mice; Figure 5 The figure is a statistical graph of the relative expression amount of IL-13 mRNA in the esophageal tissue of each group of mice. DETAILED DESCRIPTION

[0023] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present application and not to limit the present application.

[0024] The raw materials and reagents used in the present application are all conventional commercially available products unless otherwise specified; the methods used in the present application are all conventional methods in the art unless otherwise specified.

[0025] DHA and ETA were purchased from Runke Bioengineering (Fujian) Co., Ltd.; C57BL / 6 mice were provided by Sibeifu (Suzhou) Biotechnology Co., Ltd. (China), and the qualified certificate is SCXK(Su)2022-0006.

[0026] Example 1 The present embodiment provides a special medical purpose formula food composition suitable for food protein allergic infants, which comprises 70 mg of DHA, 70 mg of ETA, and 12 mg of nucleotides. The nucleotides comprise the following components in the following mass percentages: 30% of cytidine disodium, 21% of uridine disodium, 23% of adenosine monophosphate, 16% of inosine disodium, and 10% of guanosine disodium.

[0027] Example 2 The present embodiment provides a special medical purpose formula food composition suitable for food protein allergic infants, which comprises 98 mg of DHA, 107 mg of ETA, and 35 mg of nucleotides. The nucleotides comprise the following components in the following mass percentages: 36% of cytidine disodium, 25% of uridine disodium, 20% of adenosine monophosphate, 12% of inosine disodium, and 7% of guanosine disodium.

[0028] Example 3 The present embodiment provides a special medical purpose formula food composition suitable for food protein allergic infants, which comprises 200 mg of DHA, 400 mg of ETA, and 58 mg of nucleotides. The nucleotides comprise the following components in the following mass percentages: 40% of cytidine disodium, 22% of uridine disodium, 18% of adenosine monophosphate, 10% of inosine disodium, and 10% of guanosine disodium.

[0029] Comparative Example 1 The present comparative example provides a composition comprising 98 mg of DHA and 142 mg of ETA.

[0030] Comparative Example 2 The comparative example provides a composition, and the embodiment provides a composition for improving eosinophilic esophagitis, which comprises 200 mg of docosahexaenoic acid and 40 mg of nucleotides; The nucleotides comprise the following components in the following mass percentages: 36% of cytidine acid disodium, 25% of uridine acid disodium, 20% of adenosine monophosphate, 12% of inosine acid disodium, and 7% of guanosine acid disodium.

[0031] Comparative Example 3 The comparative example provides a composition, which comprises 205 mg of eicosatetraenoic acid and 35 mg of nucleotides; The nucleotides comprise the following components in the following mass percentages: 36% of cytidine acid disodium, 25% of uridine acid disodium, 20% of adenosine monophosphate, 12% of inosine acid disodium, and 7% of guanosine acid disodium.

[0032] Application Example 1. Sample Preparation Examples 1 to 3 and Comparative Examples 1 to 3 are mixed with 55.3 g of carbohydrates, 24.5 g of fats, and 13.3 g of proteins, and the energy is 2080 J. The samples are recorded as Experimental Groups 1 to 4 and Control Groups 1 to 3. The sample provided by Control Group 4 comprises 55.3 g of carbohydrates, 24.5 g of fats, and 13.3 g of proteins, and the energy is 2080 J. Vitamins and minerals are added according to the requirements of GB25596 regulations, and are consistent in the seven groups. The carbohydrates are derived from one or both of malt dextrin and high fructose corn syrup. The fats are derived from one or more of sunflower oil, low erucic rapeseed oil, medium-chain triglyceride, corn oil, and coconut oil. The proteins are derived from a compound amino acid, and the composition is as follows:

[0033] 2. Establishment of Animal Model The mice C57BL / 6 of 6 to 8 weeks old and weighing 18 to 22 g are selected for the experiment, and both males and females are acceptable. The number of mice is 10 to 15 in each group, and the mice are of clean grade or above. The mice are randomly divided into seven groups, which are recorded as Experimental Groups 1 to 3 and Control Groups 1 to 4, respectively.

[0034] The first stage sensitization stage lasts for a total of 21 days to establish a systemic immune sensitization state, and OVA and aluminum hydroxide emulsion (OVA final concentration 50 μg / each, aluminum hydroxide dose 4 mg / each) is injected intraperitoneally on days 0, 7 and 14; starting from day 28, OVA and aluminum hydroxide emulsion are injected intraperitoneally at a reduced amount every 1 day (OVA final concentration 25 μg / each, aluminum hydroxide dose 4 mg / each), to strengthen immunity, induce eosinophil infiltration and inflammatory response in the esophageal mucosa, and continue until day 63; during the challenge period (from day 28), seven groups of mice correspond to samples of experimental groups 1-3 and control groups 1-4 fed daily, and all treatment groups of mice are fed with the same amount of samples daily. All samples are collected uniformly after the end of the experiment, i.e., on day 64 (i.e., the second day after the last challenge and administration).

[0035] Effect example 1. Fecal 16S rRNA fecal intestinal flora sequencing Collection, preservation of fecal specimens and extraction of DNA: On day 64 of feeding, the feces of the seven groups of mice were collected, and before each collection, each mouse was placed in a sterilized animal cage box for 30 minutes of free activity, and about 0.2 g of feces was collected from each mouse. Bacterial DNA in the feces of experimental animals was extracted according to the QIAGEN fecal genomic DNA extraction kit instructions, and then primers 341F and 805R were used for PCR amplification in the 16S rRNA V3-V4 high variable region. After quantitative detection of the amplification product by 2% agarose gel electrophoresis, 16S rRNA gene high-throughput sequencing was performed to obtain the proportion of enterobacterium and bifidobacterium sequences.

[0036] 2. Esophageal tissue eosinophil count standard test The entire esophageal tissue of the animal was fixed with 10% neutral buffered formalin for 24-48 hours, then paraffin-embedded and made into longitudinal sections with a thickness of 4-5 μm, and specific high primary basic protein immunohistochemical staining was recommended for staining. This method can label eosinophil cytoplasmic granules through specific antibodies, and the results are accurate and reliable. When counting, at least 5 non-overlapping fields of view of the mucosal layer and submucosal layer were randomly selected by an observer who did not know the grouping information under a 400x high-power lens, and the results were expressed as "number of cells per high-power field".

[0037] 3. Esophageal epithelial thickness detection After the lower esophageal tissue was fixed with formalin and embedded in paraffin, 4 μm thick sections were prepared along the longitudinal axis, and HE staining was performed. Under a 100-fold optical microscope, 5 complete epithelial regions were randomly selected for each sample, and 3 sites were measured in each region. The vertical distance from the basal layer to the lumen was accurately calculated, and the papilla of the lamina propria was excluded to ensure accuracy. The reference value of the normal mouse esophageal epithelium was 40-50 μm, and the EoE model group usually increased to more than 80 μm. This method requires independent operation by researchers who do not know the grouping, uses vertical sections to avoid errors, and the data are expressed as "mean ± standard deviation" and statistically analyzed to evaluate the differences between groups.

[0038] 4. Detection of relative expression of IL-13 mRNA in tissues The relative expression of IL-13 mRNA in tissues was detected by real-time fluorescent quantitative PCR. About 30 mg of esophageal tissue was taken, and total RNA was extracted by the TRIzol method. After detecting the concentration and purity by a nucleic acid quantifier, 1 μg of RNA was synthesized into cDNA using a reverse transcription kit. β-actin was used as an internal reference gene, and SYBR Green fluorescent dye was used to amplify and detect in a real-time fluorescent quantitative PCR instrument. The primer sequences were designed as follows: IL-13 upstream 5'-CCTGGCTCTTGCTTGCTT-3', downstream 5'-CGTTGGGAGTCTGGTATGTA-3'; β-actin upstream 5'-GGCTGTATTCCCCTCCATCG-3', downstream 5'-CCAGTTGGTAACAATGCCATGT-3'. The reaction program included 95°C pre-denaturation for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds, and a total of 40 cycles. The relative expression of IL-13 mRNA was calculated by the 2^(-ΔΔCt) method, and the data were expressed as mean ± standard deviation, and statistically analyzed to evaluate the differences between groups.

[0039] The mice were detected according to the above method, and the specific detection results are shown in Table 1 and Table 2: Figures 1-5 and Table 1: Table 1

[0040] Note: The same letter means no significant difference, and different letters mean significant difference, P<0.05.

[0041] From Table 1 and Table 2: Figures 1-2It can be known that the enterobacter sequence proportion of the control group 4 is the highest, the enterobacter sequence proportion of the test group 3 is the lowest, the bifidobacterium sequence proportion of the control group 4 is the lowest, and the bifidobacterium sequence proportion of the test group 2 is the highest; the test proves that the composition provided by the embodiment of the present application can significantly reduce the number of enterobacter and increase the number of bifidobacterium, and the composition provided by the embodiment of the present application can be regulated by microorganisms, and effectively improve eosinophilic esophagitis.

[0042] It can be known from table 1 and Figure 3 It can be known that the esophageal tissue eosinophil count of the test groups 1-3 of the present application is significantly lower than that of the control groups 1-4, wherein the eosinophil count of the test group 2 is the lowest, the eosinophil count of the control group 4 is the highest, and is significantly higher than that of other control groups; the test proves that the composition provided by the embodiment of the present application can significantly reduce the esophageal tissue eosinophil count, and make the esophageal tissue eosinophil count return to the normal physiological range (<10 cells / HPF).

[0043] It can be known from table 1 and Figure 4 It can be known that the esophageal epithelial thickness of the test groups 1-3 of the present application is significantly lower than that of the control groups 1-4, wherein the esophageal epithelial thickness of the test group 2 is the lowest, the esophageal epithelial thickness of the control group 4 is the highest, and is significantly higher than that of other control groups; the test proves that the composition provided by the embodiment of the present application can effectively reverse esophageal epithelial hyperplasia, and make the esophageal epithelial thickness return to the normal physiological range (40-50 μm).

[0044] It can be known from table 1 and Figure 5 It can be known that the relative expression amount of IL-13 mRNA of the test groups 1-3 of the present application is significantly lower than that of the control groups 1-4, wherein the relative expression amount of IL-13 mRNA of the test group 2 is the lowest, the relative expression amount of IL-13 mRNA of the control group 4 is the highest, and is significantly higher than that of other control groups; the test proves that the composition provided by the embodiment of the present application can significantly inhibit the expression of IL-13 mRNA, and make it return to the normal level (≤1.5), and effectively correct the Th2 immune response imbalance from the molecular level.

[0045] The special medical use formula food composition suitable for food protein allergic infants provided by the embodiment of the present application can enhance the barrier function in the intestinal tract through the intestinal-esophageal axis mechanism, produce systemic anti-inflammatory mediators, and then inhibit the Th2 immune response of the whole body and the local esophagus, so that the effect of improving eosinophilic esophagitis is achieved.

[0046] The special medical use formula food composition suitable for food protein allergic infants provided by the embodiment of the present application can also improve the abundance of beneficial bacteria bifidobacterium in the intestinal tract, reduce the abundance of enterobacter, reduce the infiltration and activation of eosinophils in the esophageal tissue, improve esophageal epithelial hyperplasia for non-therapeutic purposes, reduce the levels of IL-13 and other cytokines, and thus relieve esophageal inflammation and tissue remodeling.

[0047] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A special medical purpose formula food composition suitable for infants with food protein allergies for non-therapeutic purposes of improving eosinophilic esophagitis, characterized in that, The special medical purpose formula food composition for infants with food protein allergies includes the following components: docosahexaenoic acid, eicosapentaenoic acid, and nucleotides.

2. The use as described in claim 1, characterized in that, The mass ratio of docosahexaenoic acid, eicosapentaenoic acid, and nucleotides is (0.07~0.2):(0.07~0.4):(0.012~0.058).

3. The use as described in claim 2, characterized in that, The mass ratio of docosahexaenoic acid, eicosapentaenoic acid, and nucleotides is (0.08~0.15):(0.08~0.35):(0.02~0.05).

4. The use as described in claim 3, characterized in that, The mass ratio of docosahexaenoic acid, eicosapentaenoic acid and nucleotides is (0.09~0.1):(0.09~0.2):(0.03~0.04).

5. The use as described in claim 4, characterized in that, The mass ratio of docosahexaenoic acid, eicosapentaenoic acid, and nucleotides is 0.098:0.107:0.

035.

6. The use as described in any one of claims 1 to 5, characterized in that, The nucleotide comprises the following components in the following mass percentages: 30%–40% disodium cytidine, 20%–28% disodium uridine, 18%–23% adenosine monophosphate, 10%–16% disodium inosinate, and 5%–10% disodium guanylate.

7. The use as described in claim 1, characterized in that, The special medical purpose formula food composition suitable for infants with food protein allergies is a milk protein extensively hydrolyzed formula or an amino acid formula food.

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