Application of eucommia polysaccharide obtained by ultrasonic-assisted hot extraction method in preparation of food allergy relieving product

Abstract

The invention discloses application of eucommia polysaccharide obtained by an ultrasonic-assisted hot extraction method in preparation of a product for relieving food allergy. Specifically, the application of the eucommia polysaccharide in preparation of a medicine for relieving tissue inflammation caused by food allergy, the application of the eucommia polysaccharide in preparation of a medicine for relieving intestinal tract tissue damage caused by food allergy and the application of the eucommia polysaccharide in preparation of a medicine for relieving food allergy by adjusting the structure and function of intestinal flora. The eucommia polysaccharide obtained through extraction and purification is applied to an OVA-induced mouse allergy model, a control group, an OVA sensitization group and a polysaccharide intervention group are set, and allergy symptom scores, tissue pathological changes and intestinal flora changes are evaluated. Researches prove that the eucommia polysaccharide has the effect of relieving food allergy and can be applied to preparation of products for relieving food allergy. Therefore, the invention provides a scientific basis for comprehensive utilization of eucommia ulmoides leaf resources in relieving food anaphylaxis.

Description

I. Technical Field:

[0001] This invention relates to the field of natural product application technology, specifically to the application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products to relieve food allergies. II. Background Technology:

[0002] In recent years, the incidence of food allergies has increased significantly, especially among children. Food allergies usually stem from an excessive immune response to food proteins, and clinical manifestations can range from mild skin reactions to severe anaphylactic shock. Ovalbumin (OVA)-induced allergic reactions involve the production of immunoglobulin E (IgE) antibodies and the activation of mast cells. IgE antibodies bind to IgE receptors on the surface of mast cells, prompting the release of mediators such as histamine, thereby triggering an allergic reaction. Helper T cells type 2 (Th2) are a key cellular subset for OVA-induced allergic reactions, promoting IgE production and maintaining the allergic response by secreting cytokines such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13).

[0003] Food allergies are associated with multiple factors, including genetic susceptibility, environmental factors, and alterations in the gut microbiota. In OVA-induced allergy models, gut microbiota dysbiosis has been confirmed as a key factor influencing allergic reactions. Studies have shown that the gut microbiota composition of food allergy patients differs significantly from that of healthy individuals. Gut microbiota dysbiosis may exacerbate allergic reactions, while probiotic supplementation can alleviate allergy symptoms by regulating the microbiota. Furthermore, fructooligosaccharides (FOS) have been shown to reduce OVA-induced allergic reactions by regulating gut microbiota balance and the Th17 / Treg cell ratio. A gut microbiota analysis of 226 allergic infants by Bunyavanich et al. found that Clostridium and Firmicutes were the dominant flora, and that the composition of the microbiota significantly improved food allergies. Abdel-Gadir et al. further confirmed the central role of the gut microbiota in regulating food allergy responses. The study also found that the abundance of Firmicutes in the gut of children with allergies was significantly increased, while the abundance of Bifidobacteria and Ruminococcus in the gut of children who gradually developed immune tolerance showed an upward trend. This indicates that as allergy symptoms subside, the structure and composition of the gut microbiota change, further highlighting the key role of the gut microbiota in food allergies.

[0004] Certain natural compounds (such as oleuropein) can effectively prevent and alleviate allergic reactions by enhancing the intestinal epithelial barrier function and improving the gut microbiota. Similarly, rosmarinic acid has been shown to reduce OVA-induced allergic symptoms by regulating immune responses and gut microbiota. Eucommia polysaccharides are the main active components of Eucommia ulmoides. As a green and safe natural product, they are characterized by no residues and no toxic side effects, and exhibit a variety of biological functions. Studies have shown that Eucommia polysaccharides can improve the immune function of immunosuppressed mice by regulating gut microbiota composition. However, there are currently no research reports on the effects of Eucommia polysaccharides on food allergies. III. Summary of the Invention:

[0005] The technical problem this invention aims to solve is: based on the current understanding that some natural compounds can alleviate OVA-induced allergic symptoms by regulating immune responses and gut microbiota, this invention provides a novel application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction of natural compounds in the preparation of products to alleviate food allergies. This invention applies the extracted and purified Eucommia ulmoides polysaccharide to an OVA-induced mouse allergy model, setting up a control group, an OVA-sensitized group, and a polysaccharide intervention group. Allergy symptom scores, histopathological changes, and alterations in gut microbiota are assessed. The study confirms that Eucommia ulmoides polysaccharide has an allergic effect on food allergies and can be applied in the preparation of products to alleviate food allergies. Therefore, the technical solution of this invention provides a scientific basis for the comprehensive utilization of Eucommia ulmoides leaf resources in alleviating food allergic reactions.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] This invention provides an application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products to relieve food allergies.

[0008] Based on the above-mentioned application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products for relieving food allergies, the application of Eucommia ulmoides polysaccharide in the preparation of drugs for relieving tissue inflammation caused by food allergies.

[0009] According to the above-mentioned application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products to relieve food allergies, the tissue inflammation is lymphocyte infiltration and inflammatory cell distribution in the liver, kidneys and spleen.

[0010] Based on the above-mentioned application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products for relieving food allergies, the application of Eucommia ulmoides polysaccharide in the preparation of drugs for relieving intestinal tissue damage caused by food allergies.

[0011] According to the application of Eucommia ulmoides polysaccharide obtained by the above-mentioned ultrasound-assisted thermal extraction method in the preparation of products to relieve food allergies, the intestinal tissue damage is thinning of the muscle layer, enlargement of the mucosal gap or destruction of the integrity of the epithelial tissue.

[0012] Based on the above-mentioned application of Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction in the preparation of products for relieving food allergies, the Eucommia ulmoides polysaccharide is also used in the preparation of drugs that relieve food allergies by regulating the structure and function of intestinal flora.

[0013] The application of Eucommia ulmoides polysaccharide obtained by the above-mentioned ultrasound-assisted thermal extraction method in the preparation of products for relieving food allergies is described above. The Eucommia ulmoides polysaccharide is prepared by the following ultrasound-assisted thermal extraction method:

[0014] a. Dry and pulverize Eucommia ulmoides leaves, pass them through a 60-mesh sieve, and place the resulting Eucommia ulmoides leaf powder in a Soxhlet extractor in a 45°C water bath. Reflux with petroleum ether for 6 hours to remove lipids, then allow to dry naturally. After drying, place the powder in a 90°C water bath and reflux with anhydrous ethanol for 6 hours to remove pigments and fat-soluble substances, and then allow to dry naturally.

[0015] b. Weigh 3.00g of the Eucommia ulmoides leaf powder obtained after drying in step a into an Erlenmeyer flask, add 22mL of distilled water, stir well, and place it in an ultrasonic cleaner that has been preheated to 63℃, 40KHz, and 100W for 1 hour. After ultrasonic treatment, centrifuge at 4000r / min for 15 minutes, take the supernatant and transfer it to a flask in a vacuum rotary evaporator. Set the evaporation temperature to 60℃ and concentrate the liquid. Stop the concentration when the volume is 1 / 10 of the original volume to obtain a concentrated paste.

[0016] d. Transfer the obtained concentrated paste to an Erlenmeyer flask, add anhydrous ethanol at a volume ratio of 1:4 to make the ethanol concentration in the system 80%, place the Erlenmeyer flask in a refrigerator at 4℃ for alcohol precipitation overnight, then centrifuge at 4000 r / min, take off the lower layer precipitate, and dry it in a freeze dryer to obtain crude Eucommia ulmoides polysaccharide.

[0017] e. Redissolve the obtained crude Eucommia ulmoides polysaccharide in distilled water to prepare a 6% trichloroacetic acid solution. Add the trichloroacetic acid solution to the Eucommia ulmoides polysaccharide solution at a 1:1 volume ratio, mix well, and place in a 4℃ refrigerator for alcohol precipitation overnight. Then centrifuge at 4000 r / min for 10 min. Vacuum evaporate the supernatant again and precipitate it with anhydrous ethanol at a 1:4 volume ratio overnight at 4℃. After precipitation, centrifuge at 4000 r / min for 10 min and discard the supernatant. Redissolve the precipitate in distilled water, add 2% activated carbon, and decolorize at 60℃ for 5 minutes. After 0 min, the activated carbon was removed by filtration. The polysaccharide solution was concentrated using a vacuum rotary evaporator, and anhydrous ethanol was added at a volume ratio of 1:4. The solution was then precipitated overnight at 4°C. After centrifugation, the precipitate was dissolved in distilled water and transferred to a pretreated dialysis bag for dialysis for 72 h, with water changes during the process. After dialysis, anhydrous ethanol was added to the polysaccharide solution at a volume ratio of 1:4, and the solution was precipitated overnight at 4°C. The solution was then centrifuged at 4000 r / min for 10 min, and the precipitate was freeze-dried to obtain purified Eucommia ulmoides polysaccharide.

[0018] The positive and beneficial effects of this invention are as follows:

[0019] 1. In the mouse experiment using Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction, the administration method of the present invention is gavage, the dosage is 200 mg / kg, the administration frequency is once every other day, and the administration is continued during the establishment of an allergy model in mice by oral gavage of ovalbumin (OVA).

[0020] 2. In mouse experiments using Eucommia ulmoides polysaccharides obtained by ultrasound-assisted thermal extraction, the model group mice exhibited food allergy symptoms including elevated allergy scores and a sharp drop in rectal temperature after OVA challenge. The experiments showed that Eucommia ulmoides polysaccharides significantly reduced the allergy scores of allergic mice compared to the allergy model group (P<0.01) (see appendix for details). Figure 1 It can also slow down the rate of rectal temperature decrease (see appendix for details). Figure 2 ).

[0021] 3. The technical solution of this invention was tested on mice using Eucommia ulmoides polysaccharide obtained by ultrasound-assisted thermal extraction. The results showed that Eucommia ulmoides polysaccharide could restore the morphology of liver, kidney, and spleen tissues in allergic mice and alleviate the inflammatory response (see appendix for details). Figure 3-5 Therefore, Eucommia polysaccharides can be used in the preparation of drugs to alleviate tissue inflammation caused by food allergies.

[0022] 4. During mouse experiments using Eucommia ulmoides polysaccharides obtained by ultrasound-assisted thermal extraction, the technical solution of this invention caused intestinal tissue damage, including thinning of the muscular layer, enlargement of the mucosal interstitial space, and disruption of the integrity of the epithelial tissue. Experimental results showed that Eucommia ulmoides polysaccharides, administered orally (200 mg / kg, once every other day), could reduce the interglandular spacing of the lamina propria, thicken the lamina propria, increase the number of intestinal cells, and increase the intestinal crypt depth in allergic mice (P<0.05) (see appendix for details). Figure 6 Therefore, Eucommia ulmoides polysaccharides can be used in the preparation of drugs to alleviate intestinal tissue damage caused by food allergies.

[0023] 5. In the mouse experiments using the ultrasound-assisted thermal extraction method to obtain Eucommia ulmoides polysaccharides, the polysaccharides were administered via gavage (200 mg / kg, once every other day). The results showed that Eucommia ulmoides polysaccharides could increase the Shannon index of the intestinal flora in allergic mice (see appendix for details). Figure 7 There are differences between the groups (see appendix for details). Figure 8 ); The composition of Firmicutes and Bacteroidetes at the phylum level of Eucommia polysaccharide regulation (see appendix for details). Figure 9Furthermore, the abundance of seven differentially expressed genera, including Lachnospiraceae_NK4A136_group, Oscillibacter, and Butyricicoccus, was measured at the genus level (see Appendix for details). Figure 10 Furthermore, Eucommia ulmoides polysaccharides upregulated the abundance of Oscillibacter, Butyricicoccus, and Enterobacter in the intestines of allergic mice, downregulated the abundance of Clostridium sensu stricto 1 and Turicibacter, and made Lachnospiraceae NK4A136 group the dominant bacterial genus (see appendix for details). Figure 10 Therefore, Eucommia polysaccharides can be used in the preparation of drugs to alleviate intestinal tissue damage caused by food allergies, and can also be used in the preparation of drugs to alleviate food allergies by regulating the structure and function of intestinal flora.

[0024] In summary, the present invention demonstrates through experiments that Eucommia ulmoides polysaccharides can significantly improve food allergy symptoms in mice by alleviating tissue inflammation and regulating the structure and function of intestinal flora. Furthermore, the reagents used are of clear origin and the operating procedures are standardized, indicating promising application prospects. Therefore, the Eucommia ulmoides polysaccharides obtained by the ultrasound-assisted thermal extraction method of this invention can be used in the preparation of products that alleviate food allergies. IV. Description of the attached drawings:

[0025] Figure 1 Comparison chart of allergy scores of mice in each group;

[0026] Depend on Figure 1 It can be seen that the allergy score of the OVA group was significantly higher than that of the CON group and the OVA+PS group (P<0.01), and the allergy score of the OVA+PS group was significantly higher than that of the control group (P<0.01); thus, it is indicated that Eucommia ulmoides polysaccharide can alleviate the manifestation of allergic reaction.

[0027] Figure 2 Graph showing changes in rectal temperature in each group of mice;

[0028] Depend on Figure 2 It can be seen that after OVA stimulation, the rectal temperature of mice in the OVA group dropped sharply, while the body temperature of mice in the OVA+PS group dropped more slowly than that in the model group. This indicates that allergy leads to accelerated death in mice, and Eucommia polysaccharide can delay the drop in body temperature and alleviate the rapid onset of allergy symptoms.

[0029] Figure 3 HE staining images of liver tissue from mice in each group;

[0030] Figure 4 HE staining images of mouse kidney tissue from each group;

[0031] Figure 5HE staining images of spleen tissue from mice in each group;

[0032] Depend on Figure 3-5 The results showed that the liver, kidney, and spleen of mice were stained with hematoxylin and eosin (HE) and observed. Compared with the CON group, the OVA group showed local lymphocyte infiltration and inflammatory cell distribution in the liver, kidney, and spleen. In contrast, the OVA+PS group showed normal morphology in the liver, kidney, and spleen tissues, with no obvious inflammation. These results indicate that Eucommia ulmoides polysaccharides can effectively alleviate local inflammatory responses caused by food allergies.

[0033] Figure 6 HE staining images of intestinal tissue from each group of mice;

[0034] Depend on Figure 6 It was found that the CON group had an intact epithelial structure, with tightly packed epithelial cells, intact glands, and no lesions. The OVA group showed a thinner muscular layer, increased mucosal spaces, and disruption of epithelial integrity. The OVA+PS group showed a decreased interglandular spacing in the lamina propria, a thickened lamina propria, and a significant increase in intestinal cells. This indicates that Eucommia ulmoides polysaccharides can significantly improve intestinal inflammatory responses.

[0035] Figure 7 Alpha diversity analysis of gut microbiota in each group of mice;

[0036] Depend on Figure 7 The results showed that alpha diversity analysis revealed no significant differences in the Chao1 index and good_converage index among the groups (P>0.05). However, the Simpson index in the CON group was significantly higher than that in the OVA group (P<0.05), and the Shannon index in the CON group was significantly or extremely significantly higher than that in the OVA+PS group and the OVA group (P<0.05 or P<0.01). This indicates that Eucommia polysaccharides influence microbial diversity to some extent.

[0037] Figure 8 Beta diversity analysis of gut microbiota in mice of each group;

[0038] Depend on Figure 8 The results showed that, through Beta diversity analysis of the microbial community structure and visualization using PCoA, the PC1 and PC2 factors were 11.07% and 9.6%, respectively. These results indicate that the microbial communities of the three groups exhibited significant differences, with Eucommia polysaccharides having a substantial impact on the community structure.

[0039] Figure 9 Comparison of gut microbiota composition and differences at the phylum level among different groups of mice;

[0040] Depend on Figure 9Analysis at the phylum level revealed that Firmicutes and Bacteroidota accounted for over 90% of the total gut microbiota in different treatment groups, constituting the main components of the gut microbiota. This indicates that Eucommia ulmoides polysaccharides influence the gut microbiota structure at the phylum level.

[0041] Figure 10 Comparison of gut microbiota composition and differences among different groups of mice at the genus level;

[0042] Depend on Figure 10 At the genus level, analysis revealed seven differentially expressed genera, including *Lachnospiraceae_NK4A136_group*, *Oscillibacter*, *Butyricicoccus*, *UCG-009*, *Clostridium_sensu_stricto_1*, *Enterorhabdus*, and *Turicibacter*. The expression abundance of *Oscillibacter* and *Butyricicoccus* in the CON group was significantly higher than that in the OVA group (P<0.05), but there was no significant difference compared to the OVA+PS group (P>0.05). Compared to the OVA group, *Eucommia ulmoides* polysaccharides downregulated the abundance of *Clostridium sensu stricto1* and *Turicibacter*, and upregulated the abundance of *Enterobacter*. *Lachnospiraceae_NK4A136_group* was the dominant genus in the OVA+PS group. The dominant genera in the OVA group were *Turicibacter*, *Clostridiales*, *Clostridiaceae*, *Clostridium seneu stricto*, and *Rhodospirillales*. The group with added polysaccharides showed an increase in dominant genera, including *Oscillospirales*, *Oscillospiraceae*, *Oscillibacter*, *Butyricicoccaceae*, *UCG_009*, *Coriobacterila*, *Coriobacteriales*, *Butyricicoccus*, *Eggerthellaceae*, and *Enterorhabdus*. This indicates that *Eucommia ulmoides* polysaccharides influence the gut microbiota structure at the genus level and can increase the abundance of dominant genera. V. Detailed Implementation Methods:

[0043] The present invention will be further illustrated below with reference to the embodiments, but this does not limit the scope of protection of the technical solution of the present invention.

[0044] The specific steps for detecting the effects of Eucommia ulmoides polysaccharides on the correlation between mouse gut microbiota and key gene expression are as follows:

[0045] a. Using allergic mice gavaged with 200 mg / kg Eucommia ulmoides polysaccharide as the research subjects, mesenteric lymph node tissues and intestinal flora samples were collected;

[0046] b. 16S rDNA sequencing was used to analyze the relative abundances of bacterial genera such as Blautia and Lachnospiraceae_FCS020_group in the intestinal flora.

[0047] Example 1: Application test of Eucommia ulmoides polysaccharide in relieving food allergy symptoms in BALB / c mice

[0048] 1. Preparation of experimental animals:

[0049] Thirty-six female SPF-grade BALB / c mice (6 - 8 weeks old, weighing 18 - 22 g, production license number: SCXK(Yu)2020 - 0005) were selected. The feeding environment temperature was 22 - 25°C, the humidity was 50 - 60%, with a 12h light-dark cycle, free access to food and water. The experiment started after 3 days of environmental adaptation.

[0050] 2. Grouping and administration:

[0051] The mice were randomly divided into three groups (n = 12):

[0052] Blank control group (CON group): Gavage with 0.2 mL of normal saline daily for 42 days;

[0053] Allergy model group (OVA group): Gavage with 1 mg OVA (dissolved in 0.2 mL of normal saline) on days 0, 7, 14, 21, and 28, and gavage with 0.2 mL of normal saline for the rest of the time;

[0054] Eucommia ulmoides polysaccharide group (OVA + PS group): On the basis of the OVA group treatment, an additional gavage of 200 mg / kg Eucommia ulmoides polysaccharide (dissolved in 0.2 mL of normal saline) was given every other day for 42 days.

[0055] 3. High-dose stimulation and index detection:

[0056] On the 42nd day, the OVA group and the OVA + PS group were gavaged with 5 mg OVA (dissolved in 0.2 mL of normal saline), and the CON group was gavaged with 0.2 mL of normal saline; 1 hour after stimulation, a mouse rectal thermometer (accuracy 0.1°C) was inserted about 1 cm into the rectum of the mouse to measure and record the rectal temperature; at the same time, 8 scorers independently scored according to the standard of "0 points for no symptoms - 1 point for scratching ears and nose - 2 points for eye and nose swelling / reduced activity - 3 points for asthma / dyspnea - 4 points for muscle contraction - 5 points for convulsion / death" without communication, and the average value was taken as the allergy score for each group.

[0057] 4. Results and analysis:

[0058] Allergy score: The OVA group (3.2±0.4) was extremely significantly higher than the CON group (0.1±0.1) and the OVA+PS group (1.5±0.3) (P<0.01), indicating that eucommia ulmoides polysaccharide can reduce the severity of allergy;

[0059] Rectal temperature: After stimulation in the OVA group, the body temperature decreased by 2.1±0.3°C, and in the OVA+PS group, it decreased by 1.2±0.2°C. Eucommia ulmoides polysaccharide significantly slowed down the rate of body temperature decrease (P<0.05).

[0060] The results of this experiment are shown in Appendix Figure 1 (Comparison chart of allergy scores of mice in each group) and Appendix Figure 2 (Change chart of rectal temperature of mice in each group). It can be seen from Figure 1 that: The allergy score of the OVA group was extremely significantly higher than that of the CON group and the OVA+PS group (P<0.01), and the allergy score of the OVA+PS group was extremely significantly higher than that of the control group (P<0.01); indicating that eucommia ulmoides polysaccharide can relieve the manifestation of allergic reactions. It can be seen from Figure 2 that: After OVA challenge, the rectal temperature of mice in the OVA group decreased rapidly, while the decrease in body temperature of mice in the OVA+PS group was slower than that in the model group; indicating that allergy led to an accelerated death of mice, and eucommia ulmoides polysaccharide can delay the decrease in body temperature and relieve the acute onset of allergic symptoms.

[0061] It can be seen from Example 1 that the eucommia ulmoides polysaccharide obtained by the ultrasonic-assisted hot extraction method of the present invention can be applied to the preparation of drugs for relieving tissue inflammation caused by food allergy.

[0062] Example 2: Experiment on tissue morphology observation method

[0063] 1. Preparation of experimental animals:

[0064] Select 36 female SPF-grade BALB / c mice (6-8 weeks old, weighing 18-22 g, production license number: SCXK(Yu)2020-0005), raise them in an environment with a temperature of 22-25°C, a humidity of 50-60%, a 12h light-dark cycle, free access to food and water, and start the experiment after adapting to the environment for 3 days.

[0065] 2. Grouping and administration:

[0066] Randomly divide the mice into three groups (n = 12):

[0067] Blank control group (CON group): Intragastrically administer 0.2 mL of normal saline daily for 42 days;

[0068] Allergy model group (OVA group): Intragastrically administer 1 mg of OVA (dissolved in 0.2 mL of normal saline) on days 0, 7, 14, 21, and 28, and intragastrically administer 0.2 mL of normal saline at other times;

[0069] Eucommia polysaccharide group (OVA+PS group): In addition to the OVA group treatment, 200 mg / kg Eucommia polysaccharide (dissolved in 0.2 mL physiological saline) was administered by gavage every other day for 42 days.

[0070] 3. Sample processing:

[0071] After the experiment, mice were euthanized by cervical dislocation. The liver, kidney, spleen (0.5cm×0.5cm×0.3cm each) and jejunum segment (1cm) were quickly separated, rinsed with PBS buffer (Beijing Solarbio Science & Technology Co., Ltd.), and immediately placed in 4% paraformaldehyde fixative for 24h at 4℃.

[0072] 4. Paraffin embedding and sectioning:

[0073] Dehydration: The fixed sample was placed in 70% ethanol (1h), 80% ethanol (1h), 90% ethanol (1h), 95% ethanol (twice, 30min each time), and anhydrous ethanol (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., twice, 30min each time) for dehydration in sequence.

[0074] Clearing: Transfer to xylene (Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., twice, 20 min each time) to clear;

[0075] Embedding: After the sample has become transparent, it is placed in melted paraffin (58-60℃) and immersed in the paraffin 3 times (1 hour each time). Then it is placed in a wax block mold, a label is inserted, and the wax block is removed after the paraffin has solidified.

[0076] Sectioning: Cut the wax block into 5μm thick sections using a paraffin microtome, mount them on glass slides, and bake at 60℃ for 2 hours.

[0077] 5. HE staining:

[0078] Dewaxing: The sections were placed in xylene (twice, 10 min each), anhydrous ethanol (twice, 5 min each), 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min), and then washed with distilled water (5 min).

[0079] Staining: Immerse in hematoxylin staining solution (Beijing Regen Biotechnology Co., Ltd.) for 5 min, rinse with tap water for 10 min, differentiate with 1% hydrochloric acid ethanol for 30 s, rinse with tap water for 10 min, stain with eosin staining solution (Beijing Regen Biotechnology Co., Ltd.) for 3 min, and rinse with tap water for 5 min.

[0080] Dehydration and clearing: Immerse the samples successively in 70% ethanol (30 s), 80% ethanol (30 s), 90% ethanol (30 s), 95% ethanol (twice, 1 min each), absolute ethanol (twice, 1 min each), and xylene (twice, 1 min each).

[0081] Mounting: Drop neutral balsam, cover with a coverslip, and air dry at room temperature.

[0082] 6. Observation and analysis:

[0083] Observe the sections using ECHO software in conjunction with an RVL-100-G microscope. Randomly select 5 fields of view for each group:

[0084] Liver / kidney / spleen: Local lymphocyte infiltration was visible in the OVA group. The tissue morphology of the OVA+PS group was similar to that of the CON group, with no obvious inflammation.

[0085] For the test results of this example, see Appendix Figure 3-5 , and the livers, kidneys, and spleens of the mice were stained with HE and observed. Compared with the CON group, local lymphocyte infiltration and distribution of inflammatory cells were observed in the livers, kidneys, and spleens of the mice in the OVA group. In contrast, the tissue morphology of the livers, kidneys, and spleens of the mice in the OVA+PS group was normal, and no obvious inflammation occurred. The test results showed that eucommia ulmoides polysaccharide could restore the tissue morphology of the livers, kidneys, and spleens of allergic mice and alleviate the inflammatory response. Therefore, eucommia ulmoides polysaccharide can effectively relieve the local inflammatory response caused by food allergy.

[0086] Example 3: Experiment on 16S rDNA sequencing method

[0087] 1. Preparation of experimental animals:

[0088] Select 36 female SPF-grade BALB / c mice (6-8 weeks old, weighing 18-22 g, production license number: SCXK(Yu)2020-0005). Keep them in an environment with a temperature of 22-25°C, a humidity of 50-60%, a 12-hour light-dark cycle, and free access to food and water. Start the experiment after 3 days of environmental adaptation.

[0089] 2. Grouping and administration:

[0090] Randomly divide the mice into three groups (n = 12):

[0091] Blank control group (CON group): Intragastrically administer 0.2 mL of normal saline daily for 42 days;

[0092] Allergy model group (OVA group): Intragastrically administer 1 mg of OVA (dissolved in 0.2 mL of normal saline) on days 0, 7, 14, 21, and 28, and administer 0.2 mL of normal saline for the rest of the time;

[0093] Eucommia polysaccharide group (OVA+PS group): In addition to the OVA group treatment, 200 mg / kg Eucommia polysaccharide (dissolved in 0.2 mL physiological saline) was administered by gavage every other day for 42 days.

[0094] 3. Extraction of gut microbiota DNA:

[0095] Take 0.2 g of mouse cecal contents and place them in a 2 mL centrifuge tube. Add 1 mL of PBS buffer, vortex for 10 seconds, centrifuge at 12000 rpm for 5 min at 4°C, and discard the supernatant. Add 500 μl of MagPure Soil DNALQ Kit lysis buffer and follow the kit instructions.

[0096] Add 20 μl Proteinase K, incubate at 56 °C for 30 min, add 300 μl Buffer CL, vortex for 5 s, and incubate at 70 °C for 10 min;

[0097] Add 300 μl of anhydrous ethanol, vortex for 5 s, transfer the mixture to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid.

[0098] Add 500 μl of Buffer PW1, centrifuge at 12000 rpm for 1 min, and discard the waste liquid; add 500 μl of Buffer PW2, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and repeat once;

[0099] Centrifuge at 12000 rpm for 2 min, transfer the adsorption column to a new tube, add 50 μl of Elution Buffer, incubate at room temperature for 5 min, centrifuge at 12000 rpm for 1 min to obtain DNA, and use Nanodrop to detect purity (A260 / A280 = 1.8-2.0) and concentration (≥20 ng / μl).

[0100] 4. PCR amplification and library construction:

[0101] First round of PCR: Using DNA as a template, the V3-V4 region of the 16S rRNA gene was amplified using primers 343F (5'-TACGGRAGGCAGCAG-3') and 798R (5'-AGGGTATCTAATCCT-3'). The reaction system (25 μl) consisted of: 12.5 μl of 2×Taq PlusMaster Mix, 0.5 μl each of forward and reverse primers (10 μM), 1 μl of DNA template, and 10.5 μl of ddH2O. The reaction program was: 94℃ for 5 min, 94℃ for 30 s, 56℃ for 30 s, 72℃ for 45 s for 25 cycles, and 72℃ for 10 min.

[0102] Product purification: The PCR product was purified using AMPure XP beads (1:1 volume ratio) and eluted in 20 μl ddH2O;

[0103] Second round of PCR: Barcode primers were added for library construction. The reaction volume (25 μl) was the same as in the first round. The cycle was repeated 10 times. The product was purified again with magnetic beads and quantified using Qubit (≥10 ng / μl).

[0104] Library quality control: Agilent 2100 was used to detect the size of the library fragments (400-500bp). After passing the test, the library was sent to Shanghai Ouyi Biotechnology Co., Ltd. for sequencing using the Illumina NovaSeq 6000 platform to generate 250bp paired-end reads.

[0105] 5. Bioinformatics analysis:

[0106] Data filtering: Use Cutadapt software to remove primer sequences, Trimmomatic software to filter low-quality reads (Q<20), and DADA2 software to remove chimeras, to obtain the ASV table and representative sequences;

[0107] Species annotation: Representative sequences were compared with the Silva (version 138) database using the q2-feature-classifier software to obtain species classification information;

[0108] Diversity analysis: α-diversity (Chao1, Shannon, Simpson, good_converage) was calculated using QIIME 2, and β-diversity was plotted as an unweighted Unifrac PCoA diagram using R language;

[0109] Differential analysis: Kruskal-Wallis test was used to analyze species differences between groups, and LEfSe analysis (LDA>2) was used to screen for differentially expressed bacterial genera.

[0110] For details of the results of this embodiment, please refer to the appendix. Figure 6-10 .Depend on Figure 6 It was found that the CON group had an intact epithelial structure, with tightly packed epithelial cells, intact glands, and no lesions. In the OVA group, the muscle layer was thinner, the mucosal spaces were larger, and the integrity of the epithelial tissue was disrupted. In the OVA+PS group, the interglandular spacing in the lamina propria was reduced, the lamina propria was thickened, the number of intestinal cells was significantly increased, and the intestinal inflammation was significantly improved. This indicates that Eucommia ulmoides polysaccharides can significantly improve intestinal inflammatory responses. Figure 7Alpha diversity analysis showed no significant differences in Chao1 and good_converage indices among the groups (P>0.05). The Simpson index in the CON group was significantly higher than that in the OVA group (P<0.05), and the Shannon index in the CON group was significantly or extremely significantly higher than that in the OVA+PS group and the OVA group (P<0.05 or P<0.01). This indicates that Eucommia ulmoides polysaccharides influence gut microbiota diversity to some extent. Figure 8 The results show that, through Beta diversity analysis of the microbial community structure and visualization using PCoA, the PC1 and PC2 factors were 11.07% and 9.6%, respectively. These results indicate significant differences in the microbial communities among the three groups; Eucommia ulmoides polysaccharides have a significant impact on the community structure. Figure 9 Analysis at the phylum level revealed that Firmicutes and Bacteroidota accounted for over 90% of the total gut microbiota in different treatment groups, constituting the main components of the gut microbiota; this indicates that Eucommia ulmoides polysaccharides influence the gut microbiota structure at the phylum level. Figure 10At the genus level, analysis revealed seven differentially expressed genera, including *Lachnospiraceae_NK4A136_group*, *Oscillibacter*, *Butyricicoccus*, *UCG-009*, *Clostridium_sensu_stricto_1*, *Enterorhabdus*, and *Turicibacter*. The expression abundance of *Oscillibacter* and *Butyricicoccus* in the CON group was significantly higher than that in the OVA group (P<0.05), but there was no significant difference compared to the OVA+PS group (P>0.05). Compared to the OVA group, *Eucommia ulmoides* polysaccharides downregulated the abundance of *Clostridium sensu stricto1* and *Turicibacter*, and upregulated the abundance of *Enterobacter*. *Lachnospiraceae_NK4A136_group* was the dominant genus in the OVA+PS group. The dominant genera in the OVA group were *Turicibacter*, *Clostridiales*, *Clostridiaceae*, *Clostridium seneu stricto*, and *Rhodospirillales*. The group with added polysaccharides showed an increase in dominant genera, including *Oscillospirales*, *Oscillospiraceae*, *Oscillibacter*, *Butyricicoccaceae*, *UCG_009*, *Coriobacterila*, *Coriobacteriales*, *Butyricicoccus*, *Eggerthellaceae*, and *Enterorhabdus*. This indicates that *Eucommia ulmoides* polysaccharides influence the gut microbiota structure at the genus level and can increase the abundance of dominant genera.

[0111] As can be seen from Example 3, the Eucommia ulmoides polysaccharide obtained by the ultrasound-assisted thermal extraction method of the present invention can be used in the preparation of drugs to relieve intestinal tissue damage caused by food allergies; it can also be used in the preparation of drugs to relieve food allergies by regulating the structure and function of intestinal flora.

[0112] In summary, the Eucommia ulmoides polysaccharide obtained by the ultrasound-assisted thermal extraction method of this invention can be used in the preparation of products to alleviate food allergies.

Claims

1. Use of eucommia ulmoides polysaccharide obtained by ultrasonic-assisted hot extraction in preparation of food allergy alleviating products.

2. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 1 in the preparation of food allergy relief products, characterized in that: Use of the eucommia ulmoides polysaccharide in preparation of drugs for alleviating tissue inflammation caused by food allergy.

3. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 2 in the preparation of food allergy relief products, characterized in that: The tissue inflammation is lymphocyte infiltration and inflammatory cell distribution in liver, kidney and spleen.

4. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 1 in the preparation of food allergy relief products, characterized in that: Use of the eucommia ulmoides polysaccharide in preparation of drugs for alleviating intestinal tissue damage caused by food allergy.

5. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 4 in the preparation of food allergy relief products, characterized in that: The intestinal tissue damage is thinning of muscle layer, increase of mucosa gap or destruction of epithelial tissue integrity.

6. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 1 in the preparation of food allergy relief products, characterized in that: Use of the eucommia ulmoides polysaccharide in preparation of drugs for alleviating food allergy by regulating intestinal flora structure and function.

7. The use of the ultrasonic-assisted hot extraction eucommia ulmoides polysaccharide according to claim 1 in the preparation of food allergy relief products, characterized in that, The eucommia ulmoides polysaccharide is prepared by the following ultrasonic-assisted hot extraction method: a. Dry and crush eucommia ulmoides leaves, pass through a 60-mesh sieve, and place the obtained eucommia ulmoides leaf powder in a Soxhlet extractor in a 45℃ water bath, and use petroleum ether to reflux for 6h to remove lipids, and then naturally dry; after drying, place in a 90℃ water bath to reflux in anhydrous ethanol for 6h to remove pigments and fat-soluble substances, and then naturally dry; b. Take 3.00g of the dried eucommia ulmoides leaf powder obtained in step a into a conical flask, add 22mL of distilled water, stir uniformly, and then place in an ultrasonic cleaner preheated to 63℃, 40KHz and 100w, and ultrasonically treat for 1h; after ultrasonic treatment, centrifuge at 4000r / min for 15min, take the supernatant and move to a flask matched with a vacuum rotary evaporator, set the evaporation temperature to 60℃, and perform concentration treatment; when the volume is 1 / 10 of the original volume, stop the concentration, and obtain a concentrated paste; d. Transfer the obtained concentrated paste to a conical flask, add anhydrous ethanol at a volume ratio of 1:4 to make the ethanol concentration in the system 80%, and place the conical flask in a 4℃ refrigerator for alcohol precipitation overnight, and then centrifuge at 4000r / min to take the lower layer precipitate, and dry in a freeze dryer to obtain eucommia ulmoides crude polysaccharide; e. Redissolve the obtained eucommia ulmoides crude polysaccharide in distilled water, prepare a 6% trichloroacetic acid solution, add the eucommia ulmoides crude polysaccharide aqueous solution at a volume ratio of 1:1, mix uniformly, and then place in a 4℃ refrigerator for alcohol precipitation overnight, and then centrifuge at 4000r / min for 10min; after vacuum rotary evaporation of the supernatant, precipitate the same by using anhydrous ethanol at a volume ratio of 1:4, and place in a 4℃ refrigerator overnight; after precipitation, centrifuge at 4000r / min for 10min, and discard the supernatant; Redissolve the precipitate in distilled water, add 2% activated carbon, decolorize at 60℃ for 50min, filter to remove the activated carbon, concentrate the polysaccharide solution by using a vacuum rotary evaporator, add anhydrous ethanol at a volume ratio of 1:4, and place in a 4℃ refrigerator for alcohol precipitation overnight; after centrifugation of the precipitate, dissolve in distilled water, transfer to a pretreated dialysis bag for dialysis for 72h, and change the water during the dialysis; after the dialysis is completed, add anhydrous ethanol to the polysaccharide solution at a volume ratio of 1:4, place in a 4℃ refrigerator for alcohol precipitation overnight, and then centrifuge at 4000r / min for 10min, and freeze-dry the precipitate to obtain purified eucommia ulmoides polysaccharide.

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