Application of cynomorium songaricum total polysaccharide in preparation of medicament for inhibiting parabacteroides dielsii

Abstract

The invention belongs to the technical field of natural medicines, and particularly provides an application of cynomorium songaricum total polysaccharide in any one or more of the following applications: (1) preparing a medicament for inhibiting parabacteroides dielsii P.disassonis; (2) preparing a medicament for preventing and / or treating chronic airway inflammatory diseases caused by parabacteroides dielsii; according to the invention, the relevance between the P.distasonis and the asthma is proposed for the first time, and a new target spot is provided for asthma treatment by inhibiting the P.distasonis; the new medicinal application of the cynomorium songaricum total polysaccharide is developed, the effect of the cynomorium songaricum total polysaccharide on inhibiting P.distans is utilized, the medicine capable of treating asthma is prepared, and the blank of targeted therapy of asthma aiming at the strain in the prior art is filled; the invention provides a new scheme different from the existing asthma treatment medicines, is expected to overcome the problems of systemic side effects or curative effect limitation and the like possibly caused by long-term use of the existing medicines, and provides a new choice for asthma treatment.

Description

Technical Field

[0001] This invention relates to the field of natural medicine technology, specifically to the use of total polysaccharides from Cynomorium songaricum in the preparation of agents that inhibit or treat *Pseudomonas difficile*, or in the preparation of agents that prevent and / or treat chronic airway inflammatory diseases caused by *Pseudomonas difficile*. Background Technology

[0002] 1. Properties and characteristics of Cynomorium songaricum and its active ingredients

[0003] Cynomorium songaricum Rupr. is a parasitic plant belonging to the Cynomoraceae family. Its dried, fleshy stem is used medicinally, and it is mainly produced in the arid regions of Northwest China. Traditional Chinese medicine considers it to be warm in nature and sweet in taste, entering the kidney and large intestine meridians, and possessing the effects of tonifying kidney yang, nourishing essence and blood, and moistening the intestines to relieve constipation (Pharmacopoeia of the People's Republic of China, 2025 edition). The main active components of Cynomorium songaricum include polysaccharides, flavonoids, amino acids, and trace elements, among which total polysaccharides (CSTP) are one of the key components for its pharmacological effects.

[0004] Total polysaccharides from Cynomorium songaricum are composed of various monosaccharides (such as glucose, galactose, and mannose) linked by glycosidic bonds, with molecular weights typically ranging from several thousand to tens of thousands of Da. They are characterized by good water solubility and broad bioactivity. Existing research indicates that total polysaccharides from Cynomorium songaricum possess various pharmacological effects, including immunomodulatory, antioxidant, anti-inflammatory, and anti-fatigue properties, and have shown application potential in areas such as enhancing immunity and improving gut microbiota balance.

[0005] 2. Current treatment techniques and mechanisms of action for asthma

[0006] Asthma is a chronic inflammatory airway disease whose pathogenesis involves processes such as immune cell activation, cytokine release, and airway remodeling. Current clinical treatment mainly relies on glucocorticoids (such as budesonide), β2-receptor agonists (such as salbutamol), and leukotriene modifiers. While these drugs can relieve symptoms, long-term use may lead to systemic side effects (such as hormone dependence) or limited efficacy (such as poor response in some patients with refractory asthma).

[0007] 3. Biological characteristics and current understanding of Parabacteroides distasonis

[0008] *Parabacteroides*, belonging to the genus *Parabacteroides* within the phylum Bacteroidetes, is an important component of the human gut microbiota. Its physiological functions primarily involve carbohydrate metabolism, short-chain fatty acid production, and intestinal barrier maintenance. Current research has found that *P. distasonis* possesses dual potential for both pathogenicity and prebiotic activity, with its abundance showing a significant negative correlation with disease states such as obesity, non-alcoholic fatty liver disease, and diabetes (Liu Hongwei & Liu Shuangjiang, Cell Reports (2019, 26, 222-235)). However, its abundance is increased in patients with psoriasis, neonatal cholestasis, alopecia areata, hypertension, and polycystic ovary syndrome. Nevertheless, other experimental data present contradictory results, suggesting that *Parabacteroides* may play a dual role depending on the specific circumstances.

[0009] Although total polysaccharides from Cynomorium songaricum have been shown to possess immunomodulatory activity, existing literature (such as patent CN102526046A and the journal *International Journal of Biological Macromolecules* 2020, 163:1679-1688) only reports their applications in anti-tumor or intestinal diseases. Currently, no research reports any technical solutions for inhibiting the effects of total polysaccharides from Cynomorium songaricum on this bacterium. In existing technologies, gut microbiota-targeted therapy for asthma remains limited to broad-spectrum microbiota regulation (such as probiotic mixtures), lacking precise intervention against specific strains, and new medicinal uses for total polysaccharides from Cynomorium songaricum have not yet been developed.

[0010] The purpose of this invention is to provide a novel use for total polysaccharides from *Cistanche deserticola*, specifically in the preparation of drugs for inhibiting *Parabacteroides distasonis* to treat asthma. Given that there are currently no studies reporting a link between *P. distasonis* and asthma, nor any research on the inhibition of this bacterium by total polysaccharides from *Cistanche deserticola*, this invention aims to: propose for the first time the association between *P. distasonis* and asthma, and provide a new target for asthma treatment by inhibiting this bacterium; develop a novel pharmaceutical use for total polysaccharides from *Cistanche deserticola*, utilizing its inhibitory effect on *P. distasonis* to prepare drugs capable of treating asthma, filling the gap in existing technologies for targeted asthma treatment against this strain; and provide a new approach different from existing asthma treatments (such as glucocorticoids, β2-receptor agonists, etc.), potentially overcoming the systemic side effects or efficacy limitations that may arise from long-term use of existing drugs, thus providing a new option for asthma treatment. Summary of the Invention

[0011] The purpose of this invention is to provide the use of total polysaccharides from *Cistanche deserticola* in the preparation of drugs that inhibit *Parabacteroides distasonis* for the treatment of asthma. The total polysaccharides are active ingredients extracted from the fleshy stems of *Cistanche deserticola*. After water extraction, alcohol precipitation, deproteinization, and dialysis purification, their purity is ≥86%, and their molecular weight ranges from 36464 to 24 kDa. The monosaccharide composition is mainly glucose, galactose, and mannose. This polysaccharide can specifically inhibit the growth and metabolism of *P. distasonis*, regulate the balance of intestinal flora, and thereby alleviate airway inflammation through the "gut-lung axis," thus achieving asthma treatment and solving the problems mentioned in the background art.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] This invention provides the application of total polysaccharides from Cynomorium songaricum in any one or more of the following:

[0014] (1) Preparation of agents to inhibit *Pseudomonas difficile*;

[0015] (2) Prepare agents for the prevention and / or treatment of chronic airway inflammatory diseases caused by Parabacterium difficile.

[0016] Furthermore, the preparation method of the total polysaccharide of Cynomorium songaricum includes the following steps: take dried Cynomorium songaricum fleshy stems, crush them through an 80-mesh sieve, first extract them with 95% ethanol by reflux to remove fat-soluble components, add water to the residue at a material-to-liquid ratio of 1:25 and extract 4 times at 100℃ (4 hours each time), combine the extracts, concentrate them, add 4 times the volume of 95% ethanol to precipitate polysaccharides, remove protein by papain method, purify by dialysis bag and freeze dry to obtain Cynomorium songaricum total polysaccharide powder, wherein the molecular weight range of the total polysaccharide of Cynomorium songaricum is 36464~24KDa.

[0017] Furthermore, the effective concentration of total polysaccharides from Cynomorium songaricum in the agent is 0.5-1.0 mg / mL.

[0018] Furthermore, the chronic inflammatory airway diseases include asthma.

[0019] Furthermore, the medicine is an oral preparation.

[0020] Furthermore, the pharmaceutical preparation includes pharmaceutically acceptable excipients, which are one or more of fillers, binders, disintegrants, lubricants, and flavoring agents.

[0021] The present invention also provides a composition for inhibiting *Pseudomonas diffusa*, comprising the total polysaccharides of *Cistanche deserticola* described above.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. The pro-asthmatic effect of *Pseudomonas dilataniae* was clearly identified for the first time, providing a precise target for asthma treatment.

[0024] Existing technologies have not yet found a link between *P. distasonis* and asthma. This invention, through in vivo experiments, is the first to demonstrate that *P. distasonis* colonization can significantly aggravate airway inflammation in asthmatic mice—increasing the eosinophil count in bronchoalveolar lavage fluid (BALF) by 60%-70% and the IL-4 level by 1.35 times (P<0.01). This clearly establishes a pathological link between "*P. distasonis* colonization and aggravated asthma inflammation," filling a gap in the study of the gut microbiota mechanism of asthma and providing a specific pathogenic bacterial target for targeted intervention in asthma treatment, overcoming the limitation of traditional asthma treatment lacking precise microbial targets.

[0025] 2. Total polysaccharides from Cynomorium songaricum exhibit specific in vitro inhibitory activity against *Pseudomonas difficile*, avoiding the drawbacks of broad-spectrum antibacterial activity.

[0026] This invention demonstrates through in vitro experiments that total polysaccharides from Cynomorium songaricum exhibit concentration-dependent inhibitory effects against *P. distasonis*, with a minimum inhibitory concentration (MIC) of 0.5-1.0 mg / mL. At 1.0 mg / mL, the 24-hour inhibition rate reaches 37%, while exhibiting no significant inhibitory effect on other intestinal commensal bacteria (inhibition rate <10%). This specific antibacterial effect can target and inhibit the pro-inflammatory bacterium *P. distasonis* while minimizing interference with normal intestinal flora, thus avoiding the technical drawbacks of broad-spectrum antibiotics that lead to intestinal barrier function damage and decreased flora diversity. This provides a new approach for gut microbiota-friendly asthma intervention.

[0027] 3. The technical advantages and application potential of natural product antibacterial properties

[0028] The extraction process for total polysaccharides from Cynomorium songaricum employs a water extraction and alcohol precipitation method, enabling ton-scale production (purity ≥86%). This reduces production costs compared to chemically synthesized antibacterial agents. Furthermore, based on existing publicly available technologies (see CN110721193A), it demonstrates good safety at conventional dosages with no significant toxicity risks. Simultaneously, the characteristics of natural products make them less likely to induce bacterial resistance, addressing the industry pain point of drug resistance development with long-term use of chemical antibiotics. This lays the foundation for developing drug-free adjunctive asthma treatments. Attached Figure Description

[0029] Figure 1 Establishment of an HDM-induced asthma model and drug administration protocol;

[0030] Figure 2 To validate gut microbiota colonization (****p<0.001);

[0031] Figure 3The results of pathological sections of lung tissue after HE staining;

[0032] Figure 4 The mRNA levels of IL-4, IL-5, and IL-13 in the lung tissue of mice in each group and the content of IL-4 in BALF were compared with the normal group (**p<0.01, ***p<0.005, ****p<0.001). # p<0.05, ## p<0.01 compared to the model group);

[0033] Figure 5 The results of total white blood cell count in BALF (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001 compared with the normal group); # p<0.05 compared to the model group);

[0034] Figure 6 The serum IgE levels in each group of mice were compared with those in the normal group (p<0.001). # p<0.05 compared to the model group);

[0035] Figure 7 This is a bacterial growth curve.

[0036] Figure 8 For bacterial OD 600 and pH value;

[0037] Figure 9 The content of polysaccharides and reducing sugars in the supernatant (*p<0.05). Detailed Implementation

[0038] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] In a typical embodiment of the present invention, the application of total polysaccharides from Cynomorium songaricum in any one or more of the following:

[0040] (1) Preparation of agents to inhibit *Pseudomonas difficile*;

[0041] (2) Prepare agents for the prevention and / or treatment of chronic airway inflammatory diseases caused by Parabacterium difficile.

[0042] The chronic airway inflammatory diseases mentioned include asthma.

[0043] This invention first establishes an asthma mouse model through HDM (house dust mite) induction combined with P. distasonis gavage: Six- to eight-week-old female C57BL / 6J mice were sensitized by intratracheal instillation of HDM extract (10 μg / 80 μl / mouse) on days 0, 3, 5, 10, 12, and 14 to establish a basic asthma model; simultaneously, on day 0, P. distasonis bacterial suspension (1×10⁻⁶) was administered via gavage. 9 The relative expression level of the 16S rRNA gene of this bacterium in mouse feces was detected by qPCR (CFU / mouse / day) to verify successful intestinal colonization. Pharmacodynamic evaluation showed that compared with the HDM induction group alone, P. distasonis colonization increased the eosinophil count in bronchoalveolar lavage fluid (BALF) of mice by 60%-70%, and increased the levels of IL-4, IL-5, and IL-13 mRNA by 1.35-fold, 1.32-fold, and 1.20-fold, respectively (P<0.05). Lung tissue pathology showed more significant airway mucosal edema and inflammatory cell infiltration, confirming that this bacterium can aggravate the severity of asthma and provide a precise intestinal bacterial target for asthma treatment.

[0044] In in vitro antibacterial experiments, total polysaccharides from Cynomorium songaricum (concentration 0-2.0 mg / mL) were co-cultured with *P. distasonis* in MPYG medium at 37°C for 72 hours in anaerobically. The results showed that in the blank control group, bacteria reached the peak of the logarithmic growth phase (OD) at 24 hours. 600 =0.7±0.1); In the 1.0 mg / mL Cistanche deserticola total polysaccharide treatment group, bacterial growth was significantly inhibited, the logarithmic growth phase was delayed to 24 hours, and the peak OD was 0.7±0.1. 600 The concentration was reduced to 0.4±0.08, and the antibacterial rate reached 37% after 24 hours, indicating that the total polysaccharides of Cynomorium songaricum have a concentration-dependent growth inhibitory effect on P. distasonis.

[0045] The study also found that P. distasonis can use total polysaccharides from Cynomorium songaricum as a carbon source for metabolism, with polysaccharide consumption reaching 24.6% within 48 hours, accompanied by an acid production process (pH value dropped from 7.2 to 6.8). However, total polysaccharides from Cynomorium songaricum can still exert antibacterial effects by interfering with its metabolic pathways (such as the acid production process).

[0046] Comprehensive in vitro and in vivo experiments showed that P. distasonis colonization can aggravate airway inflammation in asthmatic mice, while the total polysaccharide of Cynomorium songaricum can reduce the production of pro-inflammatory metabolites by inhibiting the in vitro growth and in vivo colonization of the bacterium, thereby reducing the level of Th2 cytokines and alleviating asthma symptoms, thus confirming its mechanism of action in treating asthma by targeting and inhibiting P. distasonis.

[0047] The preparation method of the total polysaccharide of Cynomorium songaricum includes the following steps: dried Cynomorium songaricum fleshy stems are pulverized and passed through an 80-mesh sieve. First, 95% ethanol is refluxed to remove fat-soluble components. The residue is then extracted four times (4 hours each time) at 100℃ with water at a material-to-liquid ratio of 1:25. The extracts are combined, concentrated, and then four times the volume of 95% ethanol is added to precipitate the polysaccharide. The polysaccharide is then deproteinized using papain, purified using a dialysis bag, and freeze-dried to obtain Cynomorium songaricum total polysaccharide powder. The molecular weight range of the total polysaccharide is 36464–24 kDa. Specifically, the extraction and preparation are carried out according to the invention scheme CN110721193A.

[0048] The effective concentration of total polysaccharides from Cynomorium songaricum in the medicine is 0.5-1.0 mg / mL.

[0049] The medicine is an oral preparation.

[0050] The pharmaceutical preparation includes one or more pharmaceutically acceptable excipients, such as fillers, binders, disintegrants, lubricants, and flavoring agents.

[0051] The present invention also provides a composition for inhibiting *Pseudomonas diffusa*, comprising the total polysaccharides of *Cistanche deserticola* described above.

[0052] The present invention will be further illustrated below with specific examples. These examples are only for explaining the present invention and do not limit its content.

[0053] Example 1

[0054] The exacerbation of inflammation in a mouse model of asthma by colonization of *Pseudomonas dilatatus*

[0055] 1. Experimental Materials

[0056] Animals: SPF-grade C57BL / 6J female mice (6-8 weeks old, weighing 18-20g) were purchased from Jiangsu Qinglongshan Experimental Animal Co., Ltd. and housed in a constant temperature and humidity environment with a 12-hour light-dark cycle and free access to food and water.

[0057] Strains and reagents: P. distasonis standard strain (BNCC354946), prepared into 5×10⁻⁶ strains via anaerobic culture. 9 CFU / mL bacterial suspension; house dust mite (HDM, Stallergenes Greer); sodium pentobarbital (10mg / ml, 100μl / mite).

[0058] 2. Experimental Methods

[0059] (1) Model building Figure 1 Grouping:

[0060] Sensitization phase: On days 0, 3 and 5, mice in the model group and colonization group were injected intraperitoneally with 100 μl of anesthetic. After the mice were anesthetized, 50 μl of sensitizing agent was taken and administered via nasal and tracheal instillation for dual-effect sensitization.

[0061] Challenge phase: Days 10, 12, and 14, b) Perform the same HDM airway instillation procedure on sensitized mice.

[0062] Colonization phase: Days 0-16, colonization groups (asthma + bacterial group, control + bacterial group) were administered 0.2 mL (5 × 10⁻⁶) of P. distasonis bacterial suspension daily via gavage. 9 CFU / animal; the control group was given an equal volume of physiological saline by gavage.

[0063] Group design (n=8):

[0064] Normal control group (KB): PBS gavage + normal saline nasal and tracheal instillation;

[0065] Asthma model group (M): PBS gavage + HDM nasal and tracheal instillation;

[0066] Asthma + P. distasonis colonization group (MPD): HDM nasal and tracheal drops + P. distasonis gavage;

[0067] Blank + P. distasonis colonization group (NPD): normal saline gavage + normal saline nasal and tracheal infusion + P. distasonis gavage.

[0068] (2) Sample collection and processing:

[0069] a) Serum: After fasting for 12 hours, blood was collected by enucleating the eyeballs of mice, allowing it to stand for 30 minutes, centrifuging at 3000 rpm for 20 minutes, and the serum was stored at -80℃.

[0070] b) Bronchoalveolar lavage fluid (BALF): After cervical dislocation of mice, the trachea was exposed through a transverse incision in the neck. The trachea was ligated, and an endotracheal cannula was inserted. The BALF was repeatedly irrigated three times with 0.8 mL of pre-cooled PBS to ensure a recovery rate greater than 80%. The collected BALF was centrifuged at 2000 rpm for 5 min at 4°C. The precipitate was used for cell counting, and the supernatant was used for inflammatory factor detection.

[0071] c) Lung tissue: The thoracic cavity was longitudinally dissected along the neck to expose the heart and lungs. The upper right lobe of the lung was taken and fixed in neutral tissue fixative for H&E staining and immunohistochemistry. The remaining lung tissue was stored at -80°C.

[0072] d) Colon, cecum tissue and contents: The mouse peritoneum was longitudinally dissected to expose the intestines. The entire intestinal tissue from the cecum to the anus was dissected and washed in pre-chilled PBS to remove adhering adipose tissue and mesentery. The intestinal segment was laid flat on a pre-chilled glass plate, and the contents of the colon and cecum were carefully removed. Approximately 0.5 cm of the colon was cut and placed in neutral tissue fixative for immunohistochemical analysis. The remaining intestinal segment was stored at -80°C.

[0073] (3) Detection indicators and methods:

[0074] a) Validation of gut microbiota colonization: Feces were collected from mice on day 16, and the copy number of the P. distasonis-specific 16S rRNA gene was detected by qPCR, with universal bacterial primers as internal controls (see Table 1).

[0075] Table 1 Primer Information

[0076]

[0077] b) Lung tissue pathology: HE staining was used to observe whether there was congestion, emphysema, or inflammatory cell infiltration in the alveolar walls of lung tissue sections; whether there was inflammatory cell infiltration around the blood vessels and bronchi in the lungs; the number of goblet cells in the walls of the small bronchial tubes in the lungs; and the degree of inflammatory exudation in the lumen.

[0078] c) Determination of serum IgE and BALF IL-4 levels: Perform the procedure according to the instructions using an ELISA kit to determine the serum IgE and BALF IL-4 levels.

[0079] d) Cell classification and counting in BALF: After centrifugation, the lower layer of cells from the collected BALF was added to 15 μl of PBS, gently vortexed, and 10 μl was transferred to the center of a glass slide for spotting. After air-drying in a fume hood, Wright-Giemsa staining was performed. At least 200 cells were counted under an upright microscope. Based on morphological characteristics, the cells were classified into eosinophils, alveolar macrophages, lymphocytes, and neutrophils. The types and proportions of inflammatory cells in the BALF of each group of mice were analyzed.

[0080] e) qPCR technology was used to detect the mRNA levels of IL-4, IL-5, and IL-13 in lung tissue: RNA was extracted from lung tissue, and the nucleic acid content was measured using a Nanonp80 micro spectrophotometer. The RNA dilution volume was calculated using an 800 ng loading volume. A 20 μL reaction system was prepared according to the reverse transcription kit instructions, and cDNA was obtained by reverse transcription according to the kit's reaction program. Primer sequences are shown in Table 2, and the amplification program was set according to the instructions. The final results were based on 2... -ΔΔ The CT algorithm uses β-actin as an internal reference to calculate the relative mRNA levels in each group of samples.

[0081] Table 2 Primer Information

[0082]

[0083] e) Statistical analysis was performed using SPSS software. The t-test was used to compare two independent samples. The results were expressed as mean ± standard deviation (X ± SD). *p<0.05 indicated a significant difference compared with the control group, **p<0.01 indicated an extremely significant difference compared with the control group, #p<0.05 indicated a significant difference compared with the model group, and ##p<0.01 indicated an extremely significant difference compared with the model group.

[0084] 3. Experimental Results

[0085] (1) Validation of gut microbiota colonization:

[0086] like Figure 2 As shown, the relative abundance of P. distasonis in the cecum of the MPD and NPD groups increased by 4 to 5 times, confirming successful colonization.

[0087] (2) Results of HE staining of lung tissue:

[0088] like Figure 3 As shown, HE staining of lung tissue revealed that in the blank control group mice, the bronchial and alveolar structures were clearly visible, with no inflammatory response. The model group mice, compared to the control group, exhibited a significant inflammatory response, with obvious inflammatory cell infiltration around the bronchi. The model group mice treated with *P. distasonis* showed an exacerbated inflammatory response. In contrast, the control group mice treated with *P. distasonis* did not show a significant inflammatory response, indicating that *P. distasonis* itself does not directly trigger lung inflammation.

[0089] (3) Effects of P. distasonis on Th2 cytokines in BALF and lung tissue of asthmatic mice

[0090] Th2-mediated immune inflammation is also manifested in the massive secretion of related cytokines. Therefore, the secretion of IL-4, IL-5, and IL-13 in each group of mice was detected at different levels using ELISA and qPCR techniques. Figure 4 As shown, the results indicated that *P. distasonis* intervention further aggravated HDM-induced IL-4 release, with IL-4 levels increasing by 1.35-fold (P<0.01). Simultaneously, at the transcriptional level, it promoted the mRNA expression of IL-4, IL-5, and IL-13, with IL-4, IL-5, and IL-13 mRNA levels increasing by 1.35-fold, 1.32-fold, and 1.20-fold, respectively (P<0.05). These results suggest that *P. distasonis* can exacerbate airway inflammation in asthma by stimulating the secretion of Th2 cytokines.

[0091] (4) Cell classification and counting results in BALF

[0092] like Figure 5 As shown, significant changes occurred in the distribution of leukocyte subsets in the bronchoalveolar lavage fluid (BALF) of mice in each group. Compared with the normal control group, the proportion of macrophages in the BALF of asthma model mice was significantly reduced, while the proportion of eosinophils increased by 60%–70%. These observations suggest that an abnormal immune response may be occurring in the lungs of asthmatic mice. After intervention with *P. distasonis*, the proportion of eosinophils showed an increasing trend compared with the asthma model group, which may indicate an exacerbation of asthma. In addition, the proportion of eosinophils in the blank control group also increased slightly after intervention with *P. distasonis*, although this change did not reach statistical significance.

[0093] (5) Effect of P. distasonis on serum IgE levels in asthmatic mice

[0094] like Figure 6 As shown, in this study, compared with the untreated blank control group, the serum immunoglobulin E (IgE) level in the asthma model group mice was significantly increased, and this difference was statistically significant. Further analysis revealed that after P. distasonis intervention, serum IgE levels were further increased compared with the model group, suggesting that P. distasonis may play a promoting role in the pathological process of asthma. Furthermore, even in the blank control group mice without induced asthma, P. distasonis intervention also induced an increase in serum IgE levels, indicating that P. distasonis itself may have the potential to induce an immune response.

[0095] Example 2

[0096] In vitro antibacterial experiment of total polysaccharides from Cynomorium songaricum against *Pseudomonas difficile*.

[0097] 1. Experimental Materials

[0098] Strains: P. distasonis standard strain Columbia blood plate was purchased from Henan Beina Biotechnology Testing and Inspection Co., Ltd. (BNCC354946).

[0099] Drug: Total polysaccharides of Cynomorium songaricum (purity 89%, molecular weight 36464-24KDa), extracted and prepared according to the invention scheme CN110721193A.

[0100] Culture media: MPYG medium basal, vitamin K3-heme solution, volatile fatty acid solution (all purchased from Shandong Top Biotechnology Co., Ltd.)

[0101] 2. Experimental Methods

[0102] (1) Preparation of culture medium:

[0103] Weigh 2.66g of MPYG culture base into a 250ml round-bottom flask, add 2ml of vitamin K, 2ml of volatile fatty acids, and 96ml of ultrapure water. Heat under reflux with a heating mantle until the red color fades. Aliquot the culture medium into 12ml shaker tubes, 6ml of liquid culture medium per tube, in an anaerobic environment (10% carbon dioxide, 10% hydrogen, 80% nitrogen). Sterilize in a portable pressure steam sterilizer at 121℃ for 30 minutes, then cool in an anaerobic environment for later use.

[0104] (2) Preparation of bacterial culture: After the purchased enteric bacteria strains were activated by culturing on Columbia blood agar plates for 3 days, the strains were inoculated into MPYG liquid medium. One loopful of colonies from the Columbia blood agar plate was picked up with a 20 μL sterile inoculation loop, rubbed to dissolve in the liquid medium, and cultured in an anaerobic environment at 37°C until the logarithmic growth phase.

[0105] (3) Adding total polysaccharide of Cynomorium: During the logarithmic growth phase of bacteria, add different concentrations of total polysaccharide of Cynomorium to the above bacterial solution to make the final concentration of Cynomorium polysaccharide in the liquid culture medium 0, 0.1, 0.5, 1, 2 mg / ml. Shake to dissolve fully and continue anaerobic culture.

[0106] (4) Growth curve determination: The samples were taken at 0, 12, 24, 36, 48, 60 and 72 hours after 48-72 hours of anaerobic culture at 37℃.

[0107] (5) Verification of specific antibacterial effect: To clarify the strain selectivity of the antibacterial effect of total polysaccharide of Cynomorium songaricum, the same method and concentration gradient (0, 0.1, 0.5, 1, 2 mg / ml) as the above-mentioned experiment on P. distasonis were used to investigate its effects on the growth of Eggerthella lenta, Pseudomonas denitrificans and Escherichia coli. By comparing the growth curves and inhibition rate differences of different strains, it was verified whether total polysaccharide of Cynomorium songaricum has a specific inhibitory effect on P. distasonis.

[0108] 3. Experimental Results

[0109] like Figure 7 As shown, during the 72-hour recording period, in the blank control group (0 mg / ml), P. distasonis showed a significant growth trend, with OD... 600The value continued to rise with the extension of culture time, and then tended to stabilize after 60 hours, eventually maintaining in the range of 0.6-0.7, reflecting normal proliferation activity.

[0110] In the initial stage (0-20h), the growth of the low concentration groups (0.1mg / ml and 0.5mg / ml) was not significantly different from that of the control group. However, after 20h, the growth rate of the 0.5mg / ml group was slightly slower than that of the 0.1mg / ml group. After 60h of culture, the OD of both groups... 600 The values ​​were close to those of the control group, suggesting that the low concentration of total polysaccharide from Cynomorium songaricum had a weak inhibitory effect on bacterial proliferation. As the culture progressed, the bacteria may gradually adapt to the environment and resume growth.

[0111] The high concentration groups (1 mg / ml and 2 mg / ml) showed significant antibacterial effects: the OD of the 1 mg / ml group was significantly higher. 600 The growth rate of the OD600 was consistently lower than that of the control group, with the time to reach the logarithmic growth phase delayed to 24 hours. The peak OD600 decreased to 0.4±0.08, and the inhibition rate reached 37% after 24 hours. After 40 hours, it basically entered the plateau phase, and the final value stabilized at 0.4-0.5. The growth inhibition was more obvious in the 2 mg / ml group, and the OD600 value decreased after 20 hours. 600 The value only increased slowly, never exceeding 0.3, indicating that high concentrations of Cynomorium total polysaccharide can effectively inhibit the proliferation of P. distasonis, and the higher the concentration, the stronger the inhibitory effect. Furthermore, at a concentration of 1 mg / ml, Cynomorium polysaccharide had no significant inhibitory effect on other intestinal commensal bacteria (inhibition rate <10%).

[0112] In summary, total polysaccharides from Cynomorium songaricum exhibited concentration-dependent inhibition of P. distasonis growth in vitro, with a minimum inhibitory concentration (MIC) of 0.5–1.0 mg / mL. At low concentrations (≤0.5 mg / mL), the inhibitory effect was transient and reversible, while at high concentrations (≥1 mg / mL), it significantly and persistently hindered bacterial proliferation.

[0113] Example 3

[0114] Experiment on carbon source utilization of total polysaccharides from Cynomorium songaricum by *Pseudomonas dilatatum*

[0115] 1. Experimental Materials

[0116] Strains: P. distasonis standard strain (BNCC354946), cultured under the same conditions as in Example 2.

[0117] Culture medium:

[0118] Basal culture medium: MPYG medium without total polysaccharides from Cynomorium songaricum.

[0119] Experimental group culture medium: Modified enteric microbial culture medium (MPYG), the composition and ratio of which are shown in Table 3 below. Total polysaccharides from *Cistanche deserticola* were sterilized through a 0.22 μm filter and added as the sole carbon source to the autoclaved MPYG.

[0120] Table 3. Modified MPYG culture medium formulation

[0121] Element Content (g / L) Peptone 10 Yeast Extract 10 CSP (Cistanche deserticola polysaccharide) 5 <![CDATA[L-Cysteine-HCl-H2O]]> 0.5 <![CDATA[(NH4)2SO4]]> 0.5 <![CDATA[NaHCO3]]> 0.4 NaCl 0.08 <![CDATA[K2HPO4]]> 0.04 <![CDATA[KH2PO4]]> 0.04 <![CDATA[CaCl2]]> 0.008 <![CDATA[MgSO4]]> 0.008

[0122] 2. Experimental Methods

[0123] The strain was cultured in basal medium to the logarithmic growth phase, and the cells were collected by centrifugation, washed three times with PBS, and inoculated in equal volumes into a modified medium with Cynomorium polysaccharide as the sole carbon source. The initial bacterial density was uniformly adjusted to OD0.05. 600 The concentration was approximately 0.2, and a blank control group without bacterial inoculation was also set up. After anaerobic incubation at 37℃ for 48 h, the culture medium was centrifuged at 5000 rpm for 10 minutes to collect the supernatant.

[0124] Acid production during fermentation was detected by measuring pH values ​​at 0 and 24 hours; optical density (OD) values ​​at 600 nm were measured every 6 hours. 600 The bacterial density was reflected by the phenol-sulfuric acid method; the residual sugar content of the supernatant was determined by the phenol-sulfuric acid method (490 nm wavelength, glucose standard curve quantification), and the reducing sugar content was determined by the nitro-salicylic acid method (540 nm wavelength, glucose standard curve quantification).

[0125] 3. Experimental Results

[0126] like Figure 8 As shown, during the 48-hour recording period, the OD600 in the culture system increased from 0.17 to 0.25, and the pH value decreased from 7.2 to 6.8, indicating that P. distasonis can grow and produce acid in a culture medium with total polysaccharide of Cynomorium songaricum as the sole carbon source. Figure 9 In the experimental group, the polysaccharide content decreased by 24.64% compared with the control group, confirming that P. distasonis can utilize the total polysaccharide of Cynomorium songaricum. The reducing sugar content did not increase significantly, suggesting that the total polysaccharide of Cynomorium songaricum was degraded into reducing sugar and then further metabolized by bacteria into products such as short-chain fatty acids, which is consistent with the pH decreasing trend.

[0127] Experimental results showed that P. distasonis could grow and reproduce by metabolizing total polysaccharides of Cynomorium songaricum. The polysaccharide consumption reached 24.64% within 48 hours, accompanied by acid production. This provided experimental evidence at the metabolic level for the targeted antibacterial effect of total polysaccharides of Cynomorium songaricum.

[0128] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0129] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. Application of total polysaccharides from Cynomorium songaricum in any one or more of the following: (1) Preparation of agents to inhibit *Pseudomonas difficile*; (2) Prepare agents for the prevention and / or treatment of chronic airway inflammatory diseases caused by Parabacterium difficile.

2. The application of the total polysaccharide of Cynomorium songaricum according to claim 1 in the preparation of an agent for inhibiting Parabacterium difficile, characterized in that, The method for preparing total polysaccharides from Cynomorium songaricum, The process includes the following steps: The dried fleshy stems of *Cistanche deserticola* are pulverized and passed through an 80-mesh sieve. First, reflux extraction with 95% ethanol is used to remove fat-soluble components. The residue is then extracted four times at 100℃ with water at a material-to-liquid ratio of 1:25, each time for 4 hours. The combined extracts are concentrated, and four times the volume of 95% ethanol is added to precipitate the polysaccharides. The polysaccharides are then deproteinized using papain, purified via dialysis, and freeze-dried to obtain *Cistanche deserticola* total polysaccharide powder. The molecular weight range of the total polysaccharides is 36464–24 kDa.

3. The application according to claim 1, characterized in that, The effective concentration of total polysaccharides from Cynomorium songaricum in the pharmaceutical preparation is: 0.5-1.0 mg / mL.

4. The application according to claim 1, characterized in that, The chronic airway inflammatory diseases include asthma.

5. The application according to claim 1, characterized in that, The medicine is an oral preparation.

6. The application according to claim 1, characterized in that, The pharmaceutical preparation includes pharmaceutically acceptable excipients, which are one or more of fillers, binders, disintegrants, lubricants, and flavoring agents.

7. A composition for inhibiting *Pseudomonas difficile*, characterized in that, Including total polysaccharides from Cynomorium songaricum.

Images