Application of cocklebur fruit plant exosome in treatment of allergic rhinitis
By preparing and applying Xanthium sibiricum plant exosomes with a particle size distribution of 30-150 nm, the problems of drug side effects and long treatment cycles in the treatment of allergic rhinitis have been solved, achieving safe and effective treatment of allergic rhinitis, significantly improving symptoms and enhancing drug safety.
Patent Information
- Application Number
- CN202511865424.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-02-03
AI Technical Summary
Existing treatments for allergic rhinitis have problems such as rapid symptom relief but easy relapse, drug side effects, and long treatment cycles and poor patient compliance with allergen immunotherapy, resulting in poor patient medication adherence.
Exosomes from the cocklebur plant, with a particle size distribution in the range of 30-150 nm, were prepared through enzymatic hydrolysis, centrifugation, and density gradient purification. They are used to treat allergic rhinitis by relying on their natural anti-inflammatory and anti-allergic active ingredients to regulate immune balance and reduce inflammation of the nasal mucosa.
It significantly improves allergic behavior, reduces respiratory mucosal inflammation, downregulates the expression of allergy-related molecules, enhances the bioavailability of active ingredients, improves medication safety, and avoids safety concerns associated with traditional Chinese medicine preparations.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biomedicine, and particularly relates to an application of Xanthium sibiricum plant exosome in treating allergic rhinitis. BACKGROUND
[0002] Allergic rhinitis is a non-infectious chronic inflammatory disease of nasal mucosa mediated by immunoglobulin E (IgE). According to epidemiological statistics, the global prevalence rate thereof is as high as 10%-40%, and the adult patients in China are more than 240 million. The disease has main symptoms of nasal congestion, rhinorrhea and sneezing, and can seriously reduce the life quality of patients. If not controlled in a standard way, the disease can induce complications such as asthma and sinusitis, and form a significant public health burden.
[0003] At present, the first-line clinical intervention means for allergic rhinitis mainly include drug treatment and allergen immunotherapy. The drugs such as antihistamines and nasal glucocorticoids can quickly relieve symptoms, but have a defect of “recurrence after drug withdrawal”, and long-term use can easily cause nasal dryness, bleeding or systemic side effects. The allergen immunotherapy can regulate immunity, but has a long treatment cycle, limited applicable population and a risk of inducing severe allergic reactions, resulting in poor patient compliance in general. SUMMARY
[0004] In view of this, the application provides an application of Xanthium sibiricum plant exosome in treating allergic rhinitis, so as to safely and effectively treat allergic rhinitis.
[0005] To achieve the above object, the following solutions are adopted in the application.
[0006] The application of Xanthium sibiricum plant exosome in treating allergic rhinitis.
[0007] Preferably, the Xanthium sibiricum plant exosome has a typical cup or vesicle structure, and the particle size distribution is in a range of 30-150 nm.
[0008] Preferably, the dosage of the Xanthium sibiricum plant exosome is in a range of 0.1-100 mg / kg body weight / day.
[0009] Preferably, the preparation method of the Xanthium sibiricum plant exosome comprises the following steps.
[0010] S10. Sample pretreatment: after the Xanthium sibiricum plant material is washed, dried and crushed, the material is mixed with PBS buffer and enzyme solution for enzyme hydrolysis treatment, and then centrifuged to remove tissue residue, and the supernatant is collected;
[0011] S20. Centrifugal extraction: the supernatant is sequentially subjected to low-speed centrifugation, filtration and ultracentrifugation, the precipitate is collected and resuspended with PBS to obtain crude exosome;
[0012] S30. Density gradient purification: The crude exosomes were subjected to sucrose density gradient ultracentrifugation. The liquid corresponding to the gradient band was aspirated, and after centrifugation to remove sucrose, the precipitate was resuspended in PBS and filtered to obtain the Xanthium sibiricum plant exosomes.
[0013] Preferably, in step S10, the cocklebur plant material is selected from at least one of the whole cocklebur herb, fresh fruit, or mature processed fruit.
[0014] Preferably, in step S10, the enzymatic hydrolysate contains cellulase, pectinase, and mannitol, the pH of the enzymatic hydrolysate is 5.8, the concentration of cellulase is 2%-6%, the concentration of pectinase is 1%-3%, and the concentration of mannitol is 0.4-0.8 mol / L.
[0015] Preferably, in step S10, the enzymatic hydrolysis temperature is 40-60℃, the enzymatic hydrolysis time is 4-8h, the centrifugal force after enzymatic hydrolysis is 10000-20000 × g, and the centrifugation time is 0.5-2h.
[0016] Preferably, in step S20, the centrifugation extraction includes: sequentially centrifuging at 1000-2000 × g for 5-15 min and at 1500-2500 × g for 20-40 min to remove cell debris and obtain a supernatant. After concentrating the supernatant, centrifuge at 8000-12000 × g and 3-5℃ for 50-70 min to remove large vesicles and obtain another supernatant. Filter the obtained supernatant through a 0.45 μm filter membrane, collect the filtrate, and ultracentrifuge the filtrate at 80000-120000 × g and 3-5℃ for 50-70 min. Collect the precipitate and resuspend it in PBS to obtain the crude exosome extract.
[0017] Preferably, the crude exosomes are added sequentially to a sucrose concentration gradient of 15%, 30%, 45%, and 60%, and centrifuged at 130,000-170,000 × g for 1.5-2.5 h to obtain a precipitate. The precipitate is then resuspended in PBS, and the resuspended solution is filtered through a 0.22 μm filter membrane to obtain the Xanthium sibiricum plant exosomes.
[0018] In the above-mentioned application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis, Xanthium sibiricum plant exosomes have a clear therapeutic effect on allergic rhinitis. They can improve allergic behavior, reduce respiratory mucosal inflammation, and downregulate the expression of allergy-related molecules (IgE, IL-4, and histamine). They naturally enrich the anti-inflammatory and anti-allergic active ingredients of Xanthium sibiricum itself, significantly improving the bioavailability of active ingredients. Relying on the natural active substances they carry, they exert therapeutic effects such as inhibiting histamine release, regulating immune balance, and reducing nasal mucosal inflammatory infiltration. At the same time, due to the inherent low immunogenicity and high biocompatibility of plant exosomes, they avoid the safety concerns of traditional Chinese medicine preparations and significantly improve the safety of medication. Attached Figure Description
[0019] Figure 1 Morphological images of exosomes from the plant Xanthium sibiricum, characterized by transmission electron microscopy.
[0020] Figure 2 This is a diagram showing the particle size analysis of exosomes from the Xanthium sibiricum plant.
[0021] Figure 3 The results show the BCA concentration of exosomes from Xanthium sibiricum.
[0022] Figure 4 The behavioral scores (scratching nose and sneezing) of each group of animals were used in the application of the plant exosomes (mature processed fruit) of Xanthium sibiricum in the treatment of allergic rhinitis.
[0023] Figure 5 This image shows the HE staining results of the nasal mucosa of various groups of animals in the application of Xanthium sibiricum plant exosomes (mature processed fruit) in the treatment of allergic rhinitis according to the present invention.
[0024] Figure 6 This image shows the HE staining results of the trachea of various animals in the application of Xanthium sibiricum plant exosomes (mature processed fruit) in the treatment of allergic rhinitis according to the present invention.
[0025] Figure 7 In the application of Xanthium sibiricum plant exosomes (mature processed fruit) in the treatment of allergic rhinitis, enzyme-linked immunosorbent assay (ELISA) was used to detect changes in IgE, IL-4, and HIS in animal serum. Detailed Implementation
[0026] To make the objectives and technical solutions of this invention clearer, experiments are conducted to further illustrate the content of this invention, and to further explain the technical solutions and effects of this invention. It should be noted that the inventors conducted relevant pharmacodynamic studies to demonstrate the efficacy of Xanthium sibiricum plant exosomes in treating allergic rhinitis. It should also be noted that all the following experimental studies were conducted on the basis of demonstrating drug safety, and the dosages used in the experimental studies were all within the safe dosage range. The inventors also conducted pharmacodynamic experiments on other drugs, and the results showed the same or similar effects, but due to space limitations, they are not listed here.
[0027] 1. Pharmaceutical materials
[0028] Ovalbumin, aluminum hydroxide, physiological saline, OVA solution, loratadine (all commercially available);
[0029] Exosomes from Xanthium sibiricum (prepared by the laboratory of Ningxia Medical University) were prepared as follows:
[0030] Take out the whole Xanthium sibiricum plant, fresh fruit, and mature processed fruit separately, wash them with distilled water, and dry the surface moisture with filter paper. All sampling instruments were sterilized under high temperature and high pressure. The sampling was then performed in a clean bench. Samples were pulverized, and an appropriate amount of PBS and an equal volume of enzymatic hydrolysis solution (pH 5.8, 4% cellulase, 2% pectinase, 0.6 mol / L mannitol) were added. Enzymatic hydrolysis was carried out at 50℃ for 6 hours, followed by centrifugation at 16000 × g for 1 hour to allow the tissue residue to settle. The supernatant was collected and set aside. The supernatant was transferred to a new centrifuge tube. 1000g of the supernatant was centrifuged for 10 minutes, the precipitate was discarded, and the supernatant was collected again. 2000g of the supernatant was centrifuged for 30 minutes, the precipitate was discarded, and the supernatant was collected again. This supernatant was then placed in an ultrafiltration tube and centrifuged at 3500 rpm for 30 minutes to concentrate it. Finally, it was centrifuged again at 10000 g, 4℃, for 60 minutes to remove larger vesicles. The supernatant was then filtered through a 0.45 μm filter membrane, and the filtrate was collected. Transfer the filtrate to a new centrifuge tube and centrifuge at 100,000 g for 60 min at 4 ℃ using an ultracentrifuge rotor. Remove the supernatant; resuspend in pre-cooled 1×PBS, then filter through a 0.22 μm (aqueous phase) filter using an ultracentrifuge rotor, and centrifuge again at 100,000 g for 60 min at 4 ℃. Collect the precipitate and resuspend in an appropriate amount of PBS. Perform gradient purification using 15%, 30%, 45%, and 60% sucrose. Add the resuspended drug solution to the sucrose gradient, centrifuge at 150,000 g for 2 h, collect the liquid corresponding to the gradient band, centrifuge again at 150,000 g for 1 h to remove the sucrose, collect the precipitate, resuspend in an appropriate amount of PBS, and filter through a 0.22 μm (aqueous phase) filter to obtain Xanthium sibiricum plant exosomes.
[0031] Observation of the morphology and structure of exosomes from Xanthium sibiricum: The morphology and internal structure of exosomes were observed using transmission electron microscopy (TEM). 20 μL of exosome solution was added to a copper grid for precipitation for 1 min. The precipitate was removed with filter paper, followed by negative staining with 10 μL of uranium acetate-hydrogen peroxide solution for 1 min. The precipitate was then removed with filter paper, and the mixture was allowed to air dry for 2 h. TEM imaging was performed at 80 kV to obtain the TEM imaging results, such as... Figure 1 As shown.
[0032] Particle size analysis of exosomes from Xanthium sibiricum: The particle size distribution of exosomes was measured using a particle size analyzer. Particle size was determined using dynamic light scattering. 10 μL of exosomes was taken and diluted to an appropriate concentration. After the instrument performance was tested using standards and found to be satisfactory, exosome samples were loaded. It is important to use gradient dilutions to avoid clogging the injection needle. Once the sample was analyzed, the particle size of the exosomes detected by the instrument was obtained. Results are shown below. Figure 2 As shown.
[0033] The protein concentration of exosomes from *Xanthium sibiricum* was determined using the BCA method, and the protein concentration of the sample was calculated based on the standard curve. Exosomes were rapidly lysed at 37 °C, and 5 × RIPA lysis buffer was quickly added. After mixing, the mixture was incubated on ice for 30 min, with constant mixing during incubation. A standard sample for protein concentration determination using the BCA method was prepared, and 20 μL of the sample was added to the BCA mixture. After mixing, the sample was incubated at 37 °C for 30 min. The absorbance was measured and recorded at OD562 nm using a microplate reader. The protein concentration of the sample was calculated based on the standard curve. The results are shown below. Figure 3 As shown.
[0034] Depend on Figure 1 The results showed that the exosomes obtained from mature processed fruit, fresh fruit, and whole herb of Xanthium sibiricum all exhibited typical cup-shaped or vesicle-shaped structures when observed by transmission electron microscopy, which is consistent with the morphological characteristics of plant exosomes. This indicates that exosomes can be successfully isolated from different medicinal parts (processed fruit, fresh fruit, and whole herb).
[0035] Depend on Figure 2 It can be seen that the particle size of exosomes from the three sources is mainly concentrated in the range of 30-150 nm, with concentrated peaks and no obvious impurity peaks, indicating that the particle size uniformity of the separated exosomes is good.
[0036] Depend on Figure 3 It can be seen that the concentration of plant exosome protein (total protein content) is highest in the mature processed fruit of Xanthium sibiricum, followed by fresh fruit, and lowest in the whole herb. This indicates that there are differences in the exosome protein content of different medicinal parts, and the mature processed fruit may be a better source of exosome protein.
[0037] As a preferred option, Xanthium sibiricum plant exosomes extracted from processed fruits are theoretically more likely to inherit the active ingredient spectrum after "detoxification," and may have a greater advantage in safety, better meeting the requirements of "safety, effectiveness, and quality control" in the modernization of traditional Chinese medicine.
[0038] 2. Relevant technical solutions
[0039] 2.1 Collection of laboratory animals
[0040] Forty-eight SPF-grade male Balb / c mice were selected and provided by the Experimental Animal Center of Ningxia Medical University. They were kept in an animal room with good ventilation, dryness, room temperature of 22-25℃ and humidity of 50%-70%, with free access to water and quantitative feeding of mouse feed.
[0041] 2.2 Grouping of experimental animals
[0042] Forty-eight SPF-grade male Balb / c mice were randomly divided into six groups: blank control group, allergic rhinitis model group, positive control group (3 mg / kg), low concentration exosome gavage group (2.5 mg / kg), medium concentration exosome gavage group (5 mg / kg), and high concentration exosome gavage group (10 mg / kg), with eight mice in each group.
[0043] 2.3 Model Establishment
[0044] Allergic rhinitis models were established in 6-8 week old SPF-grade female Balb / c mice, except for the blank control group, by systemic sensitization and local challenge with ovalbumin (OVA).
[0045] Intraperitoneal sensitization procedure: 100 μg ovalbumin was used as the antigen, and 2 mg aluminum hydroxide was used as the adjuvant, dissolved in 200 μl of physiological saline. Each mouse received 0.1 ml / 10 g of this solution via intraperitoneal injection. The control group mice received an equal volume of physiological saline via intraperitoneal injection. The mouse's head, neck, trunk, and tail were held and immobilized with the left hand to fully expose the abdominal skin. The needle was inserted slowly to prevent injury to internal organs. After injection, the needle was slowly withdrawn. This was repeated every three days for a total of six times (days 1, 4, 7, 10, 13, and 16) as basic sensitization.
[0046] Local challenge procedure: One week after basal sensitization, on day 23 of the experiment, nasal challenge began, marking the start of the nasal enhancement challenge phase. Except for the normal control group mice, 20 μL of OVA solution (25 mg / mL) was instilled into each nostril of the mice. The normal control group mice received an equal dose of physiological saline in each nostril. This was repeated once daily for 10 days. The mice were held and immobilized by their head, neck, trunk, and tail with the left hand. 10 μL of solution was drawn up using a pipette and slowly introduced into the nasal cavity, avoiding coughing. This was repeated once daily for a total of 10 days for local challenge.
[0047] 2.4 Administration method
[0048] During the nasal stimulation phase, medication was administered concurrently, specifically on day 23 of the experiment, 30 minutes before the nasal stimulation, once daily for 10 days.
[0049] The blank control group and the model group were given 0.1 mL / 10g (body weight) of normal saline daily; the positive control group was given loratadine 3 mg / kg by gavage daily; the exosome gavage low, medium and high dose groups were given exosomes 2.5 mg / kg, 5 mg / kg and 10 mg / kg by gavage daily, respectively.
[0050] 2.5 Behavioral Scoring
[0051] Thirty minutes after the last nasal instillation challenge, nasal symptoms, including the number of times mice scratched their noses and sneezed, were observed and recorded within 10 minutes in each group. Specific evaluation criteria are shown in Table 1, and the scoring results are as follows: Figure 4 As shown.
[0052] Table 1 Behavioral Scoring Table for AR Mice Item 1 minute 2 minutes 3 minutes Scratch nose Scratch nose Scratch several times Scratch face more than once Sneeze 1-3 times 4-10 times More than 11 times
[0053] 2.6 H&E staining
[0054] The pathological changes in the nasal mucosa and trachea of mice in each group were analyzed by H&E staining, and the results are as follows: Figure 5 and Figure 6 As shown.
[0055] 2.7 Detection of inflammatory factors
[0056] Changes in serum IL-4, HIS, and IgE were observed using enzyme-linked immunosorbent assay (ELISA).
[0057] 3. Experimental Results
[0058] Depend on Figure 4 The results showed that the scores for nose scratching and sneezing in group M (model group) were significantly higher than those in group C (normal group), indicating that the AR model was successfully constructed. The scores for nose scratching and sneezing in each treatment group (positive drug group (Y group), low concentration exosome gavage group (EL group), medium concentration exosome gavage group (EM group), and high concentration exosome gavage group (EH group)) were significantly lower than those in group M, indicating that Xanthium sibiricum exosomes can effectively improve the allergic behavior of AR animals.
[0059] Depend on Figure 5 , 6The results showed that the nasal mucosa and trachea of group M showed obvious inflammatory cell infiltration and tissue edema; the inflammatory manifestations of each treatment group were significantly reduced compared with group M, indicating that Xanthium sibiricum exosomes can relieve respiratory mucosal inflammation and damage caused by allergic rhinitis.
[0060] Depend on Figure 7 It can be seen that the serum levels of IgE, IL-4, and HIS in group M were significantly higher than those in group C, verifying the immune dysregulation characteristics of the AR model; the levels of IgE, IL-4, and HIS in each treatment group were significantly lower than those in group M, indicating that Xanthium sibiricum exosomes can exert an anti-allergic rhinitis effect by inhibiting the release of allergy mediators and downregulating Th2-type inflammatory responses.
[0061] In summary, Xanthium sibiricum plant exosomes have a clear therapeutic effect on allergic rhinitis, improving allergic behavior, reducing respiratory mucosal inflammation, and downregulating the expression of allergy-related molecules (IgE, IL-4, and histamine). They naturally enrich the anti-inflammatory and anti-allergic active ingredients of Xanthium sibiricum itself, significantly improving the bioavailability of these active ingredients. Relying on their own naturally occurring active substances, they exert therapeutic effects by inhibiting histamine release, regulating immune balance, and reducing nasal mucosal inflammation. Furthermore, the inherent low immunogenicity and high biocompatibility of plant exosomes avoid the safety concerns associated with traditional Chinese medicine preparations, significantly improving medication safety.
[0062] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions should all be covered within the scope of protection of the present invention.
Claims
1. An application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis.
2. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 1, characterized in that, The exosomes of the Xanthium plant have a typical cup-shaped or vesicle-shaped structure, with a particle size distribution in the range of 30-150 nm.
3. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 1, characterized in that, The dosage range of the Xanthium sibiricum plant exosomes is 0.1 - 100 mg / kg body weight / day.
4. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 1, characterized in that, The method for preparing the exosomes from Xanthium sibiricum includes the following steps: S10. Sample pretreatment: The Xanthium plant material was washed, dried and crushed, then mixed with PBS buffer and enzyme hydrolysate for enzymatic hydrolysis. After that, the tissue residue was removed by centrifugation and the supernatant was collected. S20. Centrifugation extraction: The supernatant was subjected to low-speed centrifugation, filtration and ultracentrifugation in sequence. The precipitate was collected and resuspended in PBS to obtain crude exosomes. S30. Density gradient purification: The crude exosomes were subjected to sucrose density gradient ultracentrifugation. The liquid corresponding to the gradient band was aspirated, and after centrifugation to remove sucrose, the precipitate was resuspended in PBS and filtered to obtain the Xanthium sibiricum plant exosomes.
5. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 4, characterized in that, In step S10, the cocklebur plant material is selected from at least one of the whole cocklebur herb, fresh fruit, or mature processed fruit.
6. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 4, characterized in that, In step S10, the enzymatic hydrolysate contains cellulase, pectinase, and mannitol. The pH of the enzymatic hydrolysate is 5.8, the concentration of cellulase is 2%-6%, the concentration of pectinase is 1%-3%, and the concentration of mannitol is 0.4-0.8 mol / L.
7. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 4, characterized in that, In step S10, the enzymatic hydrolysis temperature is 40-60℃, and the enzymatic hydrolysis time is 4-8h; the centrifugal force after enzymatic hydrolysis is 10000-20000 g, and the centrifugation time is 0.5-2h.
8. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 4, characterized in that, In step S20, the centrifugation extraction includes: sequentially centrifuging at 1000-2000 g for 5-15 min and at 1500-2500 g for 20-40 min to remove cell debris and obtain supernatant. After concentrating the supernatant, centrifuge at 8000-12000 g at 3-5℃ for 50-70 min to remove large vesicles and obtain supernatant again. Filter the obtained supernatant through a 0.45 μm filter membrane, collect the filtrate, and ultracentrifuge the filtrate at 80000-120000 g at 3-5℃ for 50-70 min. Collect the precipitate and resuspend it in PBS to obtain the crude exosome extract.
9. The application of Xanthium sibiricum plant exosomes in the treatment of allergic rhinitis according to claim 4, characterized in that, The crude exosomes were added sequentially to a sucrose concentration gradient of 15%, 30%, 45%, and 60%, and centrifuged at 130,000-170,000g for 1.5-2.5h to obtain a precipitate. The precipitate was resuspended in PBS, and the resuspended solution was filtered through a 0.22 μm filter membrane to obtain the Xanthium sibiricum plant exosomes.