Use of roseomonas mucosa exopolysaccharide in the preparation of a medicament for alleviating and / or treating allergic rhinitis

Abstract

The application discloses application of Roseomonos mucosa extracellular polysaccharide in preparation of a medicine for relieving and / or treating allergic rhinitis, wherein the Roseomonos mucosa extracellular polysaccharide is obtained by sterilizing, removing protein, precipitating with anhydrous ethanol, and separating and purifying a fermentation liquor of Roseomonos mucosa DL-1 through a DEAE ion exchange resin and a gel column, and the Roseomonos mucosa DL-1 has been preserved in the China General Microbiological Culture Collection Center, with a preservation number of CGMCC No. 25967. The Roseomonos mucosa DL-1 extracellular polysaccharide has a relieving and / or treating effect on OVA-induced allergic rhinitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of an extracellular polysaccharide of *Rosemonas mucinus* in the preparation of a medicament for relieving and / or treating allergic rhinitis. Background Technology

[0002] Inflammation is a defensive immune response produced by the host in response to inflammatory factors. Various cells in the body are recruited to the immune microenvironment to produce cytokines to eliminate the invasion of external factors and restore the body's health. However, persistent inflammation can damage the body's organs and systemic response, playing an important role in the development of many complex diseases.

[0003] Allergic rhinitis is an upper respiratory tract disease. Studies abroad have shown that 10% to 25% of the general population worldwide suffers from allergic rhinitis, making it a global health problem affecting approximately 500 million people globally, with a significant impact on public health. Although the pathology and treatment mechanisms of allergic rhinitis have been extensively studied, many aspects of it remain unclear and require further research. Allergic rhinitis is a chronic inflammatory disease primarily characterized by a Th2-type immune response, with pathological features including significant changes in mast cells and goblet cells. Specifically, mast cell aggregation is one of the main characteristics of allergic rhinitis. Mast cells release various inflammatory mediators through degranulation, which participate in the pathophysiological processes of allergic rhinitis and are key sensitizing cells in nasal mucosal pathological changes. Therefore, mast cells and goblet cells play important roles in allergic rhinitis. Mast cells participate in allergic reactions by releasing inflammatory mediators, while goblet cell proliferation reflects pathological changes in the nasal mucosa. Together, they drive the development and persistence of allergic rhinitis.

[0004] In most cases, antihistamines and intranasal corticosteroids can alleviate symptoms of allergic rhinitis. Common newer therapies include highly purified allergen extracts and purified or recombinant allergens, allergen peptides, and new adjuvants for allergen-specific immunotherapy, as well as specific monoclonal antibodies (biologics) to block the action of IgE, IL-4 / IL-5, and / or their receptors. Despite numerous studies on the treatment of allergic rhinitis, the efficacy of medications for allergic rhinitis remains limited.

[0005] Ovalbumin (OVA) is the primary method for establishing allergic rhinitis models in mice, with aluminum hydroxide serving as a crucial adjuvant. The process of allergic rhinitis involves the body's exposure to allergens. Antigen-presenting cells then take up information about the allergen, triggering an adaptive immune response. With the aid of adjuvants, intraperitoneal injection of OVA induces systemic sensitization in mice, followed by intranasal allergen stimulation to induce a localized allergic inflammatory response.

[0006] Polysaccharides are high-molecular-weight carbohydrates composed of more than 10 monosaccharides linked by glycosidic bonds. Bioactive polysaccharides possess functions such as immunomodulation, anti-tumor activity, hypoglycemic effects, anti-inflammation, and regulation of the intestinal microenvironment. Developing and utilizing suitable bioactive polysaccharides holds promise for providing new treatment options for allergic rhinitis. Summary of the Invention

[0007] Purpose of the invention: This invention provides a novel application of *Rosamolulus mucinus* extracellular polysaccharide in the preparation of drugs for treating allergic rhinitis.

[0008] The extracellular polysaccharide of *Rhodopseudomonas myxobolus* was obtained through the following steps: Activated *Rhodopseudomonas myxobolus* was inoculated into a fermentation medium and cultured. The resulting fermentation broth was sterilized, deproteinized, and precipitated with anhydrous ethanol. The broth was then purified using DEAE centrifugation with an exchange resin. The eluted fraction (0.2-0.5 mol / L NaCl) was collected and lyophilized. The elution was then performed using a gel column at a flow rate of 0.4-0.6 mL / min. The effluent was collected for 9-15 minutes to obtain an extracellular polysaccharide with a weight-average molecular weight of 3000-15000 Da. The *Rhodopseudomonas myxobolus* strain has the CGMCC No. 25967 preservation number.

[0009] The described *Roseomonas mucosa* strain, strain number DL-1, was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 26, 2022, with accession number CGMCC No. 25967. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. This strain was a symbiotic skin fungus strain screened by the inventors from healthy human skin in November 2021.

[0010] Specifically, the fermentation broth of *Rhodopseudomonas myxobolus* is prepared by the following method: *Rhodopseudomonas myxobolus* is inoculated into R2A medium and activated at 25-37℃ for 12-24 h; the activated *Rhodopseudomonas myxobolus* is then transferred to fermentation medium and cultured at 30-37℃ for 24-36 h.

[0011] The R2A culture medium consisted of: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, and pH 7.2.

[0012] Preferably, the fermentation medium is formulated as follows: carbon source 10-100 g / L, nitrogen source 1-30 g / L, inorganic salt 0.01-50 g / L, pH 5.0-9.0, and the solvent is water.

[0013] The carbon source is any combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol, starch, and molasses, preferably any combination of sucrose, glucose, lactose, citric acid, and starch; the nitrogen source is any combination of yeast extract, beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH4)2SO4, NH4Cl, (NH4)2HPO4, and NH4NO3, preferably any combination of peptone, yeast extract, corn steep liquor, (NH4)2SO4, NH4Cl, (NH4)2HPO4, and NH4NO3; the inorganic salt is any combination of sodium chloride, sulfate, phosphate, dihydrogen phosphate, dihydrogen phosphate, and hydrochloride.

[0014] In a preferred embodiment, the fermentation medium comprises 15.0-18.0 g / L tryptone, 2.0-4.0 g / L soybean peptone or soybean papain hydrolysate, 4.0-6.0 g / L sodium chloride, 2.0-3.0 g / L dipotassium hydrogen phosphate, 2.0-3.0 g / L glucose, and a pH of 7.2-7.3. More preferably, the fermentation medium comprises 17.0 g / L tryptone, 3.0 g / L soybean peptone or soybean papain hydrolysate, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.3.

[0015] In one specific embodiment, *Rhodopseudomonas myxobolus* extracellular polysaccharide was prepared by the following method: *Rhodopseudomonas myxobolus* was inoculated into 200 mL of fermentation medium and cultured at 32 °C for 24 h. After sterilization by centrifugation at 3000-5000 rpm for 10 min, half a volume of Sevage reagent was added to remove protein three times. Then, one volume of anhydrous ethanol was added, and the mixture was incubated overnight at 4 °C to obtain crude extracellular polysaccharide precipitate. The precipitate was reconstituted with deionized water and separated using a DEAE-52 separator. The 0.2 M NaCl eluent was collected and lyophilized. Then, the precipitate was separated using a gel column at a flow rate of 0.4–0.6 mL / min. The eluent was collected for 3–9 min to obtain extracellular polysaccharide with a weight-average molecular weight of 50–90 kDa. The eluent was collected for 9–15 min to obtain extracellular polysaccharide with a weight-average molecular weight of 3000–15000 Da. The *Rhodopseudomonas myxobolus* strain has the preservation number CGMCC No. 25967.

[0016] Preferably, the gel column is a Superdex 75prep grade gel column. After collecting the elution fraction with 0.2-0.5 mol / L NaCl, the fraction is lyophilized, dissolved in pure water, and then separated using the gel column. The eluent is pure water.

[0017] The extracellular polysaccharides with a weight-average molecular weight of 3000-15000 Da have a monosaccharide composition including glucose, mannose, and rhamnose, wherein the molar ratio of the above monosaccharides is 30-60:30-80:1-10.

[0018] The monosaccharide composition of extracellular polysaccharides with a weight average molecular weight of 50-90 kDa includes mannose, rhamnose, and galactose, with a molar ratio of 40-60:10-25:10-25.

[0019] This invention provides the role of extracellular polysaccharides from Rosomonas mucinosa in relieving allergic rhinitis.

[0020] The extracellular polysaccharide of *Rhodopseudomonas mucilaginosus* alleviates OVA-induced allergic rhinitis.

[0021] Specifically, the extracellular polysaccharide of *Rhodopseudomonas myxobolus* reduces the number of nasal scratchings, the increase of inflammatory cells in the nasal mucosa, the increase of goblet cells, and the increase of mast cells induced by OVA. At the same time, the extracellular polysaccharide of *Rhodopseudomonas myxobolus* inhibits the increase of serum IL-4, IL-13, IL-6, and IFN-γ induced by OVA.

[0022] Beneficial effects: (1) The present invention prepared high molecular weight extracellular polysaccharide and low molecular weight extracellular polysaccharide of Rosomonas DL-1 by fermentation separation method. At the same time, it was verified that both polysaccharides can alleviate OVA-induced allergic dermatitis. Specifically, Rosomonas extracellular polysaccharide can reduce the number of nasal scratching, increase of inflammatory cells in nasal mucosa, increase of goblet cells and increase of mast cells induced by OVA. At the same time, it can alleviate the increase of IL-4, IL-6 and IFN-γ levels in the serum of BALB / c allergic rhinitis mice induced by OVA, providing a new means for the treatment of allergic rhinitis. Attached Figure Description

[0023] Figure 1 This is the ion chromatogram of the standard sample;

[0024] Figure 2 Ion chromatograms of low molecular weight extracellular polysaccharides (A) and high molecular weight extracellular polysaccharides (B) from Rosomonas DL-1;

[0025] Figure 3 Scattering spectra of low molecular weight extracellular polysaccharide (A) and high molecular weight extracellular polysaccharide (B) samples of *Rhodopseudomonas mucilaginosa* DL-1;

[0026] Figure 4 The effect of extracellular polysaccharide of Rosomonas myxobolus DL-1 on the pathology of OVA-induced BALB / c allergic rhinitis mice;

[0027] Figure 5 The effect of extracellular polysaccharide of Rosomonas myxobolus DL-1 on scratching in OVA-induced BALB / c allergic rhinitis mice;

[0028] Figure 6 Effect of extracellular polysaccharide of Rosomonas myxobolus DL-1 on goblet cells of nasal mucosa in OVA-induced BALB / c allergic rhinitis mice;

[0029] Figure 7 Effect of extracellular polysaccharide of Rosomonas myxobolus DL-1 on mast cells of nasal mucosa in OVA-induced BALB / c allergic rhinitis mice;

[0030] Figure 8 Effects of extracellular polysaccharide of Rosomonas myxomatosa DL-1 on serum IL-4, IL-13, IL-6 and IFN-γ in OVA-induced BALB / c allergic rhinitis mice. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0032] The detailed information of *Rhodopseudomonas mucinosa* DL-1 used in the following examples is disclosed in CN116103205A.

[0033] The extracellular polysaccharide of *Rhodopseudomonas myxobolus* DL-1 and the extracellular polysaccharide of *Rhodopseudomonas myxobolus* have the same meaning in the text, and *Rhodopseudomonas myxobolus* DL-1 and *Rhodopseudomonas myxobolus* have the same meaning.

[0034] Example 1: Preparation of extracellular polysaccharide from Rosomonas DL-1.

[0035] 1) Culture of *Roseomonas myxomonas* DL-1

[0036] *Rosamol* DL-1 cultured on a 4℃ slant was activated and inoculated onto seed culture medium (activation medium) at 32℃ for 16 hours. 10% of the seed fermentation broth was then inoculated into the fermentation medium and cultured at 32℃ for 24 hours at 200 rpm to obtain the fermentation broth. The viable cell count in the fermentation broth reached 5 × 10⁻⁶. 9 CFU / mL or higher.

[0037] The activation medium is R2A medium, and its components are: yeast extract 0.5 g / L, peptone 0.5 g / L, casein hydrolysate 0.5 g / L, glucose 0.5 g / L, soluble starch 0.5 g / L, dipotassium hydrogen phosphate 0.3 g / L, anhydrous magnesium sulfate 0.024 g / L, sodium pyruvate 0.3 g / L, agar 15.0 g / L, and pH 7.2.

[0038] The fermentation medium consists of 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.3.

[0039] 2) Preparation of extracellular polysaccharides from Rosomonas DL-1

[0040] The fermentation broth obtained above was centrifuged (3000-4000 rpm, 10 min) for sterilization. One volume of the fermentation broth was added to two volumes of Sevag reagent (chloroform: n-butanol = 4:1), repeated 2-3 times to remove protein. Then, one volume of anhydrous ethanol was added, and the mixture was centrifuged overnight at 4°C (3000-4000 rpm, 10 min) to obtain a crude extracellular polysaccharide precipitate. After drying, the precipitate was reconstituted with deionized water. Approximately 5-6 g of the crude extracellular polysaccharide was purified using the sulfuric acid-phenol method via DEAE centrifugation and resin exchange. The 0.2 M NaCl eluent was collected, lyophilized, and dissolved in pure water to a concentration of 50 mg / ml. The precipitate was then passed through a Superdex 75 prep grade gel column (26 mm × 1000 mm, Cytiva, USA) according to the elution time to separate high molecular weight extracellular polysaccharides (3-9 min) and low molecular weight extracellular polysaccharides (9-15 min). Their monosaccharide composition and molecular weight were determined.

[0041] The method for determining the monosaccharides of extracellular polysaccharides is as follows: Using a Thermo ICS5000 ion chromatography system (ICS5000, ThermoFisher Scientific, USA), an electrochemical detector was used to analyze and detect the monosaccharide components of the mixed standards and samples. After accurately weighing the required standards for this project, water was added to prepare a 10 mg / mL standard solution stock solution. Then, appropriate amounts of the stock solution were mixed to prepare standard mixed standards with maximum index concentrations of 60 μg / mL, 50 μg / mL, or 40 μg / mL. The required series of standards were prepared according to the following concentration gradients for instrumentation.

[0042] Table 1. Monosaccharide mixed standard concentration information

[0043]

[0044]

[0045] *Standard products are mainly sourced from Sigma.

[0046] Using Dionex TM C Allergic Rhinitis boPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume: 5μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0047] The results of the standard test are as follows Figure 1 As shown, all 13 standards exhibited single peaks. The chromatogram of the low molecular weight extracellular polysaccharide sample is shown below. Figure 2 As shown in Figure A, calculations based on the standard curves of standard retention time and sample concentration determined the main monosaccharide composition of low molecular weight extracellular polysaccharides to be glucose:mannose:rhamnose = 46.00:52.15:1.84. The results for high molecular weight polysaccharides are as follows... Figure 2 As shown in B, the high molecular weight extracellular polysaccharide monosaccharides are determined to mainly include mannose, rhamnose, and glucose, in a molar ratio of 44.71:22.16:21.24.

[0048] The molecular weight of the obtained polysaccharides was further determined. The method for determining the molecular weight of extracellular polysaccharides is as follows: The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system, the liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt technology, CA, USA). Size exclusion columns (Ohpak SB-805HQ (300×8 mm), Ohpak SB-804HQ (300×8 mm), and Ohpak SB-803HQ (300×8 mm) in series) were used. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.4 mL / min, the elution gradient was isocratic, and the time was 100 min. The results are as follows: Figure 3 As shown in Figure A, the weight-average molecular weight of the low molecular weight extracellular polysaccharide EPS from *Roseola mucosa* is 11.7 kDa. Figure 3As shown in B, the weight-average molecular weight of the high molecular weight extracellular polysaccharide EPS from Rosomonas mucilage is 97.2 kDa.

[0049] Example 2: Effects of extracellular polysaccharide of *Roseola myxoma* DL-1 on nasal tissue of OVA-induced BALB / c allergic rhinitis mice.

[0050] Housing environment: SPF grade, mice: BALB / c female mice, 8 weeks old, weighing 18-20g. The preparation of the OVA-induced allergic rhinitis model includes the following steps: (1) Systemic basal sensitization: On days 0, 3, 6, 9, 12 and 15, 200μL saline containing 100μg OVA (Sigma-Aldrich, USA) and 2mg aluminum hydroxide was injected intraperitoneally; (2) Nasal enhancement challenge: On days 21-30, 10μL physiological saline containing 200μg OVA was used for intranasal treatment. After one week of stable housing, the purchased mice were randomly divided into 4 groups, with 6 biological replicates for each treatment, for a total of 24 mice. The drug administration was uniformly localized, administered in the morning and modeled in the afternoon. The treatment methods for each group are as follows:

[0051] Treatment 1 (HC) and no treatment constitute the healthy control group;

[0052] Treatment 2 (Vehicle): OVA modeling, 10 μL of physiological saline was administered via nasal drops, which constituted the model group;

[0053] Treatment 3 (DLEPS-H): Starting from day 21 of OVA modeling, 10 μL of physiological saline containing 0.5 wt% of the high molecular weight Rosomonas mucinous polysaccharide prepared in Example 1 was administered as nasal drops, which is the high molecular weight polysaccharide treatment group.

[0054] Treatment 4 (DLEPS-L): Starting from day 21 of OVA modeling, 10 μL of physiological water containing 0.5 wt% of low molecular weight Rosomonas mucinous exopolysaccharide prepared in Example 1 was administered as nasal drops, which is the low molecular weight polysaccharide treatment group.

[0055] Other routine management procedures remained consistent across all four treatments. After the experiment, tissue and serum samples were collected from the sacrificed mice. Pathological analysis of the mouse nasal mucosa was performed, and the results are as follows: Figure 4 As shown, the nasal mucosa of mice in the blank control group was intact, with no obvious inflammatory cell infiltration in the submucosa and no significant interstitial hyperplasia or edema. In the model group, the nasal mucosa epithelium was significantly damaged, with disordered cilia, a large number of neutrophils infiltrating the submucosa, and interstitial hyperplasia and edema, along with small blood vessel dilation and proliferation. Both the low-molecular-weight and high-molecular-weight extracellular polysaccharide treatment groups showed significant improvement compared to the model group, with reduced inflammatory cell infiltration and epidermal cell thickness in the mouse nasal mucosa.

[0056] Example 3: Effect of extracellular polysaccharide of *Rhodopseudomonas mucinosa* DL-1 on the number of scratches caused by nasal itching in OVA-induced BALB / c allergic rhinitis mice.

[0057] Fifteen days after OVA induction, mice were challenged by nasal drops. Twenty-four hours before sacrifice, activity was recorded for each mouse for half an hour in an undisturbed environment. The number of scratches was counted during the midpoint of the time interval. The results are as follows: Figure 5 As shown, extracellular polysaccharides from *Roseola myxoma* DL-1 had a certain alleviating effect on OVA-induced nasal itching in mice. Both the low-molecular-weight and high-molecular-weight extracellular polysaccharide treatment groups showed statistically significant differences compared to the model group. The difference between the low-molecular-weight extracellular polysaccharide treatment group and the model group was more significant (P≤0.001); the statistically significant difference between the high-molecular-weight extracellular polysaccharide treatment group and the model group was P≤0.01.

[0058] Example 4: Effect of extracellular polysaccharide of *Rhodopseudomonas mucosa* DL-1 on the number of goblet cells in the nasal mucosa of OVA-induced BALB / c mice.

[0059] The specific steps for goblet cell staining are as follows:

[0060] (1) Place paraffin sections of mouse nasal mucosa in an oven and bake at 60°C for 1-2 hours;

[0061] (2) Paraffin sections were routinely dewaxed with xylene and ethanol until hydrated;

[0062] (3) Oxidize with 0.5% periodic acid aqueous solution for 5 minutes.

[0063] (4) Rinse with distilled water for at least 5 minutes;

[0064] (5) Let the Scheffer reagent act for 10-30 minutes;

[0065] (6) Wash three times with sulfurous acid solution, each time for 1.5-2 minutes.

[0066] (7) Rinse under running water for 15 minutes;

[0067] (8) Lightly stain cell nuclei with harrihiazine for 1-2 minutes (too dark staining may indicate differentiation);

[0068] (9) Rinse with running water;

[0069] (10) Dehydrate, clear, and seal as usual.

[0070] Under a microscope, the goblet cells appeared reddish-purple, and the nuclei appeared blue. The results were as follows: Figure 6 As shown, OVA can induce the proliferation of goblet cells in the nasal mucosa of BALB / c mice. The order of proliferation is: model group, high molecular weight extracellular polysaccharide group, and low molecular weight extracellular polysaccharide group. All extracellular polysaccharide treatment groups have a significant effect on reducing the proliferation of goblet cells in the nasal mucosa of BALB / c mice induced by OVA.

[0071] Example 5: Effect of extracellular polysaccharide of *Roseola myxoma* DL-1 on mast cell infiltration in the nasal mucosa of OVA-induced BALB / c allergic rhinitis mice.

[0072] After decalcification of the heads of the sacrificed mice, they were embedded in paraffin and sectioned. Mast cells were stained with toluidine blue. The staining method was as follows: (1) The tissue was fixed in neutral formaldehyde solution or formaldehyde ethanol solution; (2) The tissue sections were dewaxed; (3) The sections were stained with toluidine blue solution (0.5g toluidine blue diluted with distilled water to 100ml) for 30min; (4) The sections were rinsed slightly with water; (5) The sections were differentiated with glacial acetic acid solution (0.5ml glacial acetic acid diluted with distilled water to 100ml) until the nuclei and granules were clear (controlled under a microscope); (6) The sections were rinsed slightly with water and dried with cold air; (7) The sections were mounted with xylene or clear neutral resin.

[0073] The results are as follows Figure 7 As shown, mast cell granules appear reddish-purple with blue nuclei. OVA treatment significantly induced mast cell infiltration in the nasal mucosa of mice. Compared with the model group, the treatment groups with *Rhodopseudomonas myxobolus* DL-1 extracellular polysaccharide could alleviate OVA-induced mast cell infiltration in mice, and the differences were significant under microscopic observation. Among them, the low molecular weight extracellular polysaccharide treatment group was more effective than the high molecular weight extracellular polysaccharide treatment group.

[0074] Example 6: Effects of extracellular polysaccharide of *Roseola myxoma* DL-1 on serum IL-4, IL-13, IL-6 and IFN-γ in OVA-induced BALB / c allergic rhinitis-like mice.

[0075] The specific experimental steps are as follows:

[0076] ① Before starting the experiment, the collected specimens and various reagents in the kit need to be placed at room temperature for 30 minutes.

[0077] ② Divide the ELISA plate into three parts: blank wells, standard wells, and sample wells. Add sample diluent, standard, and sample to be tested sequentially to each well, 100 μL per well. When adding samples, avoid touching the well walls; add the sample to the bottom of the plate and gently shake to mix, avoiding air bubbles. The sample addition should be completed within 10 minutes. Then cover the ELISA plate with a membrane and incubate it in a thermostatic water bath at 37°C for 120 minutes.

[0078] ③ During incubation, prepare the working solution A. After incubation, discard the solution and dry the plate without washing it. Add 100 μL of the working solution A to each well of the plate, shake to mix, cover the plate with a membrane, and incubate again in a thermostatic water bath at 37°C for 60 minutes.

[0079] ④ During the second incubation, dilute the concentrated washing buffer to 20 times with distilled water. Shake off the liquid in each well, add approximately 350 μL of the prepared washing buffer to each well, soak for 1-2 minutes, and then shake off the liquid in the microplate. Repeat this washing step 3 times.

[0080] ⑤ Prepare the working solution B. After washing, add about 100 μl of the working solution B to each well of the microplate, shake to mix, cover the microplate with a membrane, and incubate again in a thermostatic water bath at 37°C for 60 minutes.

[0081] ⑥ After the second incubation, discard the liquid and spin dry, then wash the plate a total of 5 times, using the same washing method as in step ④.

[0082] ⑦ Add 90 μL of substrate solution to each well of the microplate, cover with a membrane, and incubate at 37°C in the dark for about 15 minutes. When a clear gradient appears in the standard wells, stop the color development.

[0083] ⑧ Add 50 μL of stop solution to each well to terminate the reaction. Add the stop solution in the order of substrate solution addition. The reaction is terminated when the color of the liquid in the well of the ELISA plate immediately changes from blue to yellow.

[0084] ⑨ After terminating the reaction, immediately place the ELISA plate in the microplate reader, using the blank wells as controls. Set the label to 0 and the wavelength to 450 nm, and measure the OD value of each well. Then, establish a standard curve according to the instructions, generate a standard equation, and substitute the absorbance values ​​of each well into the formula of the equation to calculate the concentrations of IL-4, IL-13, IL-6, and IFN-γ in each sample.

[0085] The results are as follows Figure 8 As shown, extracellular polysaccharides of *Rosamol* DL-1 inhibited the increase of serum IL-4, IL-13, IL-6 and IFN-γ in OVA-induced allergic rhinitis-like mice. Both low-molecular-weight and high-molecular-weight extracellular polysaccharide treatment groups showed statistically significant differences compared with the model group. However, low-molecular-weight DLEPS could not significantly inhibit the increase of serum IL-13 induced by OVA, which may be due to differences between groups.

[0086] In summary, this invention provides a method for preparing and applying extracellular polysaccharide of *Roseomonas mucosa* DL-1, a dermal commensal bacterium that can alleviate allergic rhinitis. The OVA-induced BALB / c allergic rhinitis animal model verified the alleviating effect of *Roseomonas mucosa* DL-1 extracellular polysaccharide on scratching frequency, inflammatory factor accumulation, and nasal mucosal damage in mice with allergic rhinitis. Therefore, *Roseomonas mucosa* DL-1 extracellular polysaccharide has certain application prospects in the treatment of allergic rhinitis.

[0087] This invention provides a method and approach for alleviating allergic rhinitis. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. The application of *Rosedomonas mucinosa* extracellular polysaccharide in the preparation of drugs for relieving and / or treating allergic rhinitis, wherein, The extracellular polysaccharide of *Rhodopseudomonas myxobolus* was obtained through the following steps: Activated *Rhodopseudomonas myxobolus* was inoculated into a fermentation medium and cultured. The resulting fermentation broth was sterilized, deproteinized, and precipitated with anhydrous ethanol. It was then purified by DEAE centrifugation with an exchange resin. The eluted fraction (0.2-0.5 mol / L NaCl) was collected and lyophilized. The elution was then performed using a gel column at a flow rate of 0.4-0.6 mL / min. The effluent was collected for 9-15 min to obtain an extracellular polysaccharide with a weight-average molecular weight of 3000-15000 Da. The preservation number of *Rhodopseudomonas myxobolus* is CGMCC No. 25967.

2. The application according to claim 1, characterized in that, The gel column is a Superdex 75 prep grade gel column. After collecting the elution fraction with 0.2-0.5 mol / L NaCl, the fraction is lyophilized, dissolved in pure water, and then separated using the gel column. The eluent is pure water.

3. The application according to claim 1, characterized in that, The monosaccharide composition of the extracellular polysaccharide of *Rhodopseudomonas mucilaginosa* includes glucose, mannose, and rhamnose, wherein the molar ratio of the above monosaccharides is 30-60 : 30-80 : 1-10.

4. The application of *Rosemonas mucinus* extracellular polysaccharides in the preparation of drugs for relieving and / or treating allergic rhinitis, wherein, The extracellular polysaccharide of *Roseomonas myxobolus* was obtained through the following steps: Activated *Roseomonas myxobolus* was inoculated into a fermentation medium and cultured. The resulting fermentation broth was sterilized, deproteinized, and precipitated with anhydrous ethanol. It was then purified by DEAE centrifugation with an exchange resin. The eluted fraction was collected in 0.2-0.5 mol / L NaCl, lyophilized, and then separated by gel column chromatography at a flow rate of 0.4-0.6 mL / min. The effluent was collected for 3-9 min. The preservation number of *Roseomonas myxobolus* is CGMCC No. 25967.

5. The application according to claim 4, characterized in that, The gel column is a Superdex 75 prep grade gel column. After collecting the elution fraction with 0.2-0.5 mol / L NaCl, the fraction is lyophilized, dissolved in pure water, and then separated using the gel column. The eluent is pure water.

6. The application according to claim 4, characterized in that, The monosaccharide composition of the extracellular polysaccharide of *Rhodopseudomonas mucilaginosa* includes a molar ratio of mannose, rhamnose, and glucose of 40-60: 10-25: 10-25.

7. The application according to any one of claims 1 to 6, characterized in that, The fermentation medium consists of: 15.0-18.0 g / L tryptone, 2.0-4.0 g / L soybean peptone, 4.0-6.0 g / L sodium chloride, 2.0-3.0 g / L dipotassium hydrogen phosphate, 2.0-3.0 g / L glucose, and a pH of 7.2-7.

3.

8. The application according to claim 7, characterized in that, The fermentation medium consisted of 17.0 g / L tryptone, 3.0 g / L soybean peptone, 5.0 g / L sodium chloride, 2.5 g / L dipotassium hydrogen phosphate, 2.5 g / L glucose, and a pH of 7.

3.

9. The application according to claim 1 or 4, characterized in that, The extracellular polysaccharide of *Rhodopseudomonas mucilaginosus* alleviates OVA-induced allergic rhinitis.

10. The application according to claim 1 or 4, characterized in that, The extracellular polysaccharide of *Rhodopseudomonas mucosae* reduced OVA-induced nasal scratching frequency, nasal mucosal inflammatory cell increase, goblet cell increase, and mast cell increase.

11. The application according to any one of claims 1 or 3, wherein the application is characterized in that, The extracellular polysaccharide of *Rhodopseudomonas mucilaginosa* inhibited the OVA-induced increase in serum IL-4, IL-13, IL-6, and IFN-γ.

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