Compound bergamot nasal cavity lotion and application thereof

Abstract

The invention discloses a compound bergamot nasal cavity lotion and application thereof, and belongs to the technical field of medicines. According to the invention, the bergamotin, the sodium pyruvate, the honeysuckle flower extract and the lophatherum gracile polysaccharide are combined, the bergamotin can inhibit the combination of virus spike protein and ACE2, the sodium pyruvate can block a virus ROS signal channel, and the honeysuckle flower extract can resist inflammation and inhibit bacteria, through the combination of the substances, the dual mechanisms of physical flushing and drug permeation are realized, and the application prospect is wide. The obtained compound bergamot nasal cavity lotion achieves the synergistic effect, and the antiviral and anti-inflammatory effects are remarkably superior to those of a single-component lotion.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to Compound Bergamot Nasal Wash and its application. Background Technology

[0002] Upper respiratory tract infection is a common infectious disease mainly caused by respiratory viruses. It is usually caused by bacteria, viruses or fungi, leading to an inflammatory reaction of the upper respiratory tract mucosa, causing symptoms such as runny nose, nasal congestion, nasopharyngeal pain, burning and stinging sensation, etc. Some are contagious.

[0003] Nasal irrigation is a common adjunctive treatment for upper respiratory tract infections. Cleansing the nasal cavity with saline solution can effectively remove pathogens and secretions, alleviate symptoms, and promote recovery. However, while saline irrigation can improve nasal mucus clearance, it cannot effectively control bacterial and fungal infections; it only relieves symptoms and does not address the underlying cause. Traditional nasal irrigation methods, although able to alleviate upper respiratory tract infection symptoms to some extent, cannot cure the disease and have certain limitations and side effects.

[0004] In summary, there is an urgent need to provide a new type of nasal wash that can more effectively control infection, alleviate symptoms, and avoid side effects. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a compound bergamot nasal wash, comprising, by weight, the following components:

[0007] Bergamotin 1%–2%,

[0008] Sodium pyruvate 0.5%–1.2%,

[0009] Honeysuckle extract 1.5%–3%,

[0010] Nano-encapsulated Lophatherum gracile polysaccharides 0.3%–0.8%,

[0011] Imperata cylindrica root extract 0.5%–1.5%,

[0012] pH-responsive liposomes: 5%–10%

[0013] The remainder is physiological saline.

[0014] In one embodiment, the compound bergamot nasal wash comprises, by weight, the following components:

[0015] Bergamotin 1.5%,

[0016] Sodium pyruvate 0.8%,

[0017] Honeysuckle extract 2%,

[0018] Nano-encapsulated 0.5% of bamboo leaf polysaccharide

[0019] 1% Imperata cylindrica root extract

[0020] pH-responsive liposomes 5%,

[0021] The remainder is physiological saline.

[0022] In one embodiment, the chlorogenic acid content of the honeysuckle extract is ≥30%.

[0023] In one embodiment, the preparation method of the nano-encapsulated bamboo leaf polysaccharide includes: mixing chitosan and bamboo leaf polysaccharide, dissolving, homogenizing, and freeze-drying to obtain the nano-encapsulated bamboo leaf polysaccharide.

[0024] In one embodiment, the mass ratio of chitosan to Lophatherum gracile polysaccharide is 1:(1-5). Optionally, the mass ratio is 1:2.

[0025] In one embodiment, the nano-encapsulated bamboo leaf polysaccharide has a particle size of 80–150 nm.

[0026] In one embodiment, the encapsulation efficiency of the nano-encapsulated Lophatherum gracile polysaccharide is ≥85%.

[0027] In one embodiment, the pH-responsive liposome of the present invention is a liposome carrier capable of responding to the acidic environment of the nasal cavity (pH 6.0-6.5) and releasing active ingredients.

[0028] In one embodiment, the method for preparing the pH-responsive liposomes includes: mixing DPPC (dispalmitoylphosphatidylcholine), cholesterol and polyethylene glycol-polylactic acid, dissolving, evaporating the solvent to form a film, ultrasonically hydrating, and extruding to granulate, thereby obtaining the pH-responsive liposomes.

[0029] In one embodiment, the mass ratio of DPPC, cholesterol, and polyethylene glycol-polylactic acid is (5-10):(1-5):1. Optionally, the mass ratio is 7:2:1.

[0030] In one embodiment, the pH-responsive liposomes have a particle size of 80–150 nm.

[0031] The pH-responsive liposomes of this invention can precisely release active ingredients in the acidic environment of the nasal cavity (pH 6.0-6.5), with a drug release rate of ≥90% and a bioavailability increased by 2 times (compared to ordinary dosage forms).

[0032] In a second aspect, the present invention provides a method for preparing a compound bergamot nasal wash, comprising the following steps:

[0033] By weight, 1%–2% bergamot, 0.5%–1.5% Imperata cylindrica root extract, and 5%–10% pH-responsive liposomes were dissolved in an organic solvent. After removing the solvent, a lipid membrane was obtained.

[0034] Sodium pyruvate 0.5%–1.2%, honeysuckle extract 1.5%–3%, and nano-encapsulated bamboo leaf polysaccharide 0.3%–0.8% were dissolved in water to obtain a hydration medium. The hydration medium was mixed with the lipid membrane, sonicated, and extruded to obtain a drug-loaded liposome concentrate.

[0035] The pH of the drug-loaded liposome concentrate was adjusted to 6.0–6.5, and the solution was filtered to remove bacteria, thus obtaining the compound bergamot nasal wash.

[0036] In one embodiment, the pH-responsive liposome comprises DPPC, cholesterol, and polyethylene glycol-polylactic acid. Optionally, the mass ratio of DPPC, cholesterol, and polyethylene glycol-polylactic acid is (5-10):(1-5):1. More preferably, the mass ratio is 7:2:1.

[0037] In a third aspect, the present invention provides a medicine comprising the compound bergamot nasal wash described in the first aspect and pharmaceutically permissible excipients.

[0038] In a fourth aspect, the present invention also provides the use of the compound bergamot nasal wash described in the first aspect in the preparation of a medicament for treating respiratory infections and / or respiratory inflammation.

[0039] In one implementation, the respiratory infection includes SARS-CoV-2 virus infection.

[0040] In one embodiment, the respiratory infection includes an upper respiratory tract infection.

[0041] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) This invention combines bergamot extract, sodium pyruvate, honeysuckle extract and bamboo leaf polysaccharide. Bergamot extract can inhibit the binding of viral spike protein to ACE2, sodium pyruvate can block the viral ROS signaling pathway, and honeysuckle extract can have anti-inflammatory and antibacterial effects. This invention achieves a dual mechanism of "physical flushing + drug penetration" through the combination of the above substances, which can reduce viral load. The resulting compound bergamot nasal wash achieves a synergistic effect, and its antiviral and anti-inflammatory effects are significantly better than those of single-component washes.

[0044] (2) This invention uses pH-responsive liposome technology: it accurately releases active ingredients in the acidic environment of the nasal cavity (pH 6.0-6.5), with a drug release rate of ≥90% and a bioavailability that is 2 times higher than that of ordinary dosage forms.

[0045] (3) This invention uses sodium pyruvate as a tension modifier to maintain the isotonicity of the washing solution (osmotic pressure 280-320 mOsm / kg), reduce mucus irritation (score reduced by 30%), and prolong the drug retention time in mucus to 6 hours. At the same time, sodium pyruvate acts as an antioxidant to reduce the oxidative degradation rate of the formulation and improve stability.

[0046] (4) Standardized process: Bergamot extract is purified using high-speed countercurrent chromatography (purity ≥95%) and high-pressure homogenized nano-encapsulation technology (particle size 80-150nm). The process parameters are clearly defined (pressure 25MPa, temperature 45℃), making it suitable for large-scale production (batch capacity 1000L). Sodium pyruvate can be directly sourced from pharmaceutical-grade raw materials, eliminating the need for complex synthesis and reducing production costs (cost is 20% lower than traditional antiviral washes).

[0047] (5) Comprehensive indications: It is applicable to acute and chronic rhinitis, upper respiratory tract infection, allergic rhinitis, pediatric adenoid hypertrophy and respiratory viral infection, and has unique value in nasal prevention for close contacts of COVID-19 (reducing viral load by 85% after exposure). Attached Figure Description

[0048] Figure 1 To test the antiviral efficacy of bergamot nasal rinse solution.

[0049] Figure 2 The study aimed to detect the anti-inflammatory efficacy of bergamot nasal irrigation solution. In this study, A, B, and C represent the inhibitory effects of the compound lotion (bergamot compound irrigation solution) on the expression of inflammatory factors TNF-α, IL-1β, and IL-6 in nasal mucosal organoids, respectively.

[0050] Figure 3 Safety testing of bergamot extract compound lotion.

[0051] Figure 4 The viral RNA inhibition effect of compound bergamot nasal wash.

[0052] Figure 5 This study investigated the effects of Compound Bergamot Nasal Wash on inflammatory damage to nasal mucosal organoids. A, B, and C represent the effects of Compound Bergamot Nasal Wash on the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6, respectively.

[0053] Figure 6 This study investigated the effects of different pH values ​​of Compound Bergamot Nasal Wash on inflammatory damage to nasal mucosal organoids. A, B, and C represent the effects of Compound Bergamot Nasal Wash on the expression of inflammatory cytokines TNF-α, IL-1β, and IL-6, respectively. Detailed Implementation

[0054] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation of the invention in any way.

[0055] In this invention, the term "about" or "approximately" should be understood to include all values ​​within the permissible range of measurement error.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] In the following examples, the extracts of honeysuckle, bamboo leaf polysaccharide, and Imperata cylindrica root were derived from bergamot extract (CAS: 7380-40-7):

[0058] This product was prepared in-house using bergamot extract (Citrus medica 'Fingered'), a plant belonging to the Rutaceae family and the Citrus genus. The bergamot peel was washed with water, pulverized, and dried. Crude extraction was then performed using organic solvents (methanol, ethanol, ethyl acetate, etc.) at 60℃ for 8-20 minutes. The crude extract was then preliminarily separated using silica gel column chromatography and further purified using high-speed countercurrent chromatography to obtain bergamot extract with a purity of over 95%.

[0059] Honeysuckle extract (CAS: 84603-62-3):

[0060] This product is prepared in-house using fresh or dried honeysuckle (Lonicera japonica Thunb.) buds, extracted with solvents such as water and ethanol, followed by filtration, concentration, and drying to form a solid or liquid extract. Extraction methods include lime sulfur extraction, ultrafiltration, ethanol reflux extraction, and macroporous resin adsorption treatment.

[0061] Lophatherum gracile polysaccharide:

[0062] The polysaccharide was prepared in-house using dried stems and leaves of the grass Lophatherum gracile Brongn. The process involved water extraction (using ultrasonic and / or high-temperature methods), filtration, concentration, and ethanol precipitation. The polysaccharide was then washed multiple times with anhydrous ethanol, acetone, and ether, followed by vacuum drying at 30-60°C. Further protein elution and purification with macroporous resin were then followed by concentration and drying to obtain solid Lophatherum gracile polysaccharide.

[0063] Imperata cylindrica root extract:

[0064] The extract was prepared in-house using the rhizomes of Imperata cylindrica Beauv. var. major (Nees) CE Hubb., a plant of the Poaceae family. After drying, the extract was extracted by ultrasonic soaking in water, followed by filtration, concentration, and drying to obtain a solid extract.

[0065] Example:

[0066] Example 1: Preparation of Compound Bergamot Nasal Wash: Formula 1

[0067] 1) The raw materials and equipment used in the preparation are shown in Table 1.

[0068] Table 1. Raw materials and equipment used in the preparation of Compound Bergamot Nasal Wash

[0069]

[0070] 2) The preparation process is as follows:

[0071] Step 1: Preparation of nano-encapsulated bamboo leaf polysaccharides

[0072] 1.1 Dissolution: Dissolve 5g of Lophatherum gracile polysaccharide and 10g of chitosan (degree of deacetylation ≥90%) in 5ml of 1% acetic acid solution (pH 4.5) and stir until completely dissolved.

[0073] 1.2 High-pressure homogenization: The solution obtained in step 2.1 was homogenized using an APV-2000 homogenizer (1500 bar, 3 cycles) to obtain a nano suspension.

[0074] 1.3 Freeze-drying: The obtained nano suspension was pre-frozen at -50℃ for 24 hours and then vacuum dried (0.1 mbar, 48 hours) to obtain nano-encapsulated bamboo leaf polysaccharide powder.

[0075] Step 2: Preparation of pH-responsive liposomes

[0076] 2.1. Preparation of liposome membrane: Bergamotin (1.5g), Imperata cylindrica root extract (1.0g), DPPC (4g), cholesterol (3.6g), and polyethylene glycol-polylactic acid (0.4g) were dissolved in chloroform (200ml) and rotary evaporated (40℃, 200rpm) to form a uniform lipid membrane.

[0077] 2.2. Hydration and drug loading: Honeysuckle extract, Lophatherum gracile polysaccharide and sodium pyruvate were dissolved in 200ml of physiological saline to form a hydration medium. The hydration medium was added to the liposome membrane and ultrasonically treated in a 50℃ constant temperature water bath (300W, pulse mode, 5s / 5s) for 30 minutes to form a crude suspension of drug-loaded liposomes.

[0078] 2.3 Preparation of liposome microspheres: The crude suspension was circulated and extruded 5 times through a Lipex extruder (filtered through a 0.22 μm polycarbonate membrane at 50 °C) to obtain a concentrated drug-loaded liposome solution with a diameter of 100 ± 20 nm.

[0079] Step 3: Final formulation mixing and filling

[0080] 3.1 Preparation of nasal wash solution: Take the liposome concentrate, add 0.9% sterile saline, and bring the volume to 1000 ml. Stir and mix (200 rpm, 15 minutes). Adjust the pH of the mixture to 6.0–6.5 with citrate-disodium hydrogen phosphate buffer.

[0081] 3.2 Sterile filtration and filling: After filtration through a 0.22μm sterile filter membrane, the contents are dispensed into sterile nasal irrigation bottles (10ml brown glass bottles equipped with a metered spray pump), with a filling accuracy of ±1% (10.0±0.2ml per bottle).

[0082] 3) The key process control points (CPPs) and quality inspection (CQA) in the above preparation process are shown in Table 2 below:

[0083] Table 2 Key process control points and quality inspection in the preparation process.

[0084]

[0085] 4) Batch production records and process verification:

[0086] Batch size: 1000L / batch (100,000 bottles).

[0087] Process validation: Three consecutive batches of production were conducted, and the intra-batch RSD of key parameters (particle size, pH, content) was ≤2%, and the inter-batch RSD was ≤5%.

[0088] Stability data: Accelerated test (40℃, RH75%) for 6 months, bergamot retention ≥90%, no degradation of sodium pyruvate.

[0089] 5) Risk control and deviation handling

[0090] Particle size exceeds limit: If the liposome particle size is >120nm in step 2.3, it needs to be re-extruded (≤3 times) or the hydration temperature needs to be adjusted (increased to 55℃).

[0091] pH deviation: If the pH exceeds the range of 6.0 to 6.5 in step 3.1, add citric acid / sodium citrate buffer to adjust it, and retest until it is within the acceptable range.

[0092] Microbial contamination: If the sterility test is positive, the entire batch should be discarded, and the cleanliness of the sterilization equipment and environment (≥ISO 7 level) should be investigated.

[0093] Example 2: Preparation of Compound Bergamot Nasal Wash: Formula 2

[0094] 1) The raw materials and equipment used in the preparation are shown in Table 3.

[0095] Table 3. Raw materials and equipment used in the preparation of compound bergamot nasal wash.

[0096]

[0097]

[0098] 2) The preparation process is as follows:

[0099] Step 1: Preparation of nano-encapsulated bamboo leaf polysaccharides

[0100] 1.1 Dissolution: Dissolve 8g of Lophatherum gracile polysaccharide and 16g of chitosan (degree of deacetylation ≥90%) in 8ml of 1% acetic acid solution (pH 4.5) and stir until completely dissolved.

[0101] 1.2 High-pressure homogenization: The solution obtained in step 2.1 was homogenized using an APV-2000 homogenizer (1500 bar, 3 cycles) to obtain a nano suspension.

[0102] 1.3 Freeze-drying: The obtained nano suspension was pre-frozen at -50℃ for 24 hours and then vacuum dried (0.1 mbar, 48 hours) to obtain nano-encapsulated bamboo leaf polysaccharide powder.

[0103] Step 2: Preparation of pH-responsive liposomes

[0104] 2.1. Preparation of liposome membrane: Bergamotin (1.0g), Imperata cylindrica root extract (0.8g), DPPC (4g), cholesterol (3.6g), and polyethylene glycol-polylactic acid (0.4g) were dissolved in chloroform (200ml) and rotary evaporated (40℃, 200rpm) to form a uniform lipid membrane.

[0105] 2.2. Hydration and drug loading: Honeysuckle extract, Lophatherum gracile polysaccharide and sodium pyruvate were dissolved in 200ml of physiological saline to form a hydration medium. The hydration medium was added to the liposome membrane and ultrasonically treated in a 50℃ constant temperature water bath (300W, pulse mode, 5s / 5s) for 30 minutes to form a crude suspension of liposomes.

[0106] 2.3 Preparation of liposome microspheres: The crude suspension was circulated and extruded 5 times through a Lipex extruder (filtered through a 0.22 μm polycarbonate membrane at 50 °C) to obtain a concentrated drug-loaded liposome solution with a diameter of 100 ± 20 nm.

[0107] Step 3: Final formulation mixing and filling

[0108] 3.1 Preparation of nasal wash solution: Take the liposome concentrate, add 0.9% sterile saline, and bring the volume to 1000 ml. Stir and mix (200 rpm, 15 minutes). Adjust the pH of the mixture to 6.0–6.5 with citrate-disodium hydrogen phosphate buffer.

[0109] 3.2 Sterile filtration and filling: After filtration through a 0.22μm sterile filter membrane, the contents are dispensed into sterile nasal irrigation bottles (10ml brown glass bottles equipped with a metered spray pump), with a filling accuracy of ±1% (10.0±0.2ml per bottle).

[0110] 3) The key process control points (CPPs) and quality inspection (CQA) in the above preparation process are shown in Table 4 below:

[0111] Table 4 Key process control points and quality inspection in the preparation process.

[0112]

[0113]

[0114] 4) Batch production records and process verification:

[0115] Batch size: 1000L / batch (100,000 bottles).

[0116] Process validation: Three consecutive batches of production were conducted, and the intra-batch RSD of key parameters (particle size, pH, content) was ≤2%, and the inter-batch RSD was ≤5%.

[0117] Stability data: Accelerated test (40℃, RH75%) for 6 months, bergamot retention ≥90%, no degradation of sodium pyruvate.

[0118] 5) Risk control and deviation handling

[0119] Particle size exceeds limit: If the liposome particle size is >120nm in step 2.3, it needs to be re-extruded (≤3 times) or the hydration temperature needs to be adjusted (increased to 55℃).

[0120] pH deviation: If the pH exceeds the range of 6.0 to 6.5 in step 3.1, add citric acid / sodium citrate buffer to adjust it, and retest until it is within the acceptable range.

[0121] Microbial contamination: If the sterility test is positive, the entire batch should be discarded, and the cleanliness of the sterilization equipment and environment (≥ISO 7 level) should be investigated.

[0122] Example 3: Therapeutic efficacy trial of antiviral effects

[0123] Subject Sources: Patients were collected from Nanfang Hospital, Southern Medical University, between October 2020 and June 2023, who were pathologically diagnosed with nasal polyps and bullous middle turbinates after surgery, requiring surgical removal of the affected tissue. Residual tissue from fresh olfactory epithelial endoscopic surgery was also collected. All patients included in the study had signed informed consent forms (ICF) approved by the IRB / IEC before surgery. The ethics of this research project were approved by the Ethics Committee of Nanfang Hospital.

[0124] Experimental methods and efficacy indicators:

[0125] Nasal mucosal organoids were infected with SARS-CoV-2 virus (2019-nCoV-WIV04, B.1.1.7) to assess the antiviral efficacy of compound bergamot nasal wash on nasal mucosal organoids. The viral copy number in infected nasal mucosal organoids was analyzed using RT-qPCR.

[0126] RT-qPCR primers:

[0127] Forward primer:5'-GACCCCAAAATCAGCGAAAT-3'

[0128] Reverse primer:5'-TCTGGTTACTGCCAGTTGAATCTG-3'

[0129] Dosing regimens for the experimental and control groups:

[0130] Experimental group 1: 10% compound nasal wash formula 1, 90% physiological saline;

[0131] Experimental group 2: 50% compound nasal wash formula 1, 50% physiological saline;

[0132] Experimental group 3: 100% compound nasal wash formula 1

[0133] Experimental group 4: 10% compound nasal wash formula 2, 90% physiological saline;

[0134] Experimental group 5: 50% compound nasal wash formula 2, 50% physiological saline;

[0135] Experimental group 6: 100% compound nasal wash formula 2

[0136] Control group 1: 100% saline

[0137] Control group 2: 5% pH-sensitive liposome carrier, 95% physiological saline

[0138] Result: As Figure 1 As shown, Compound Bergamot Nasal Wash can inhibit SARS-CoV-2 infection in nasal mucosal organoids in a dose-dependent manner, and Formula 1 has more significant antiviral activity compared with Formula 2, proving that it has an inhibitory effect on novel coronavirus infection in nasal mucosal organoids.

[0139] Example 4: Therapeutic efficacy test of anti-inflammatory effects

[0140] Subject source: Same as Example 3

[0141] Experimental methods and efficacy indicators:

[0142] The effects of compound bergamot nasal wash on a nasal mucosa organoid model treated with LPS (1 μg / ml) were compared. Changes in the secretion levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected, and the anti-inflammatory and antioxidant effects of bergamot extract were also examined.

[0143] Experimental method: ELISA enzyme-linked detection method

[0144] Grouping and dosing regimen:

[0145] Experimental group 1: 10% compound nasal wash formula 1, 90% physiological saline;

[0146] Experimental group 2: 50% compound nasal wash formula 1, 50% physiological saline;

[0147] Experimental group 3: 100% compound nasal wash formula 1

[0148] Experimental group 4: 10% compound nasal wash formula 2, 90% physiological saline;

[0149] Experimental group 5: 50% compound nasal wash formula 2, 50% physiological saline;

[0150] Experimental group 6: 100% compound nasal wash formula 2

[0151] Control group 1: 100% saline

[0152] Control group 2: 5% pH-sensitive liposome carrier, 95% physiological saline

[0153] Test kits used:

[0154] Human TNF-α ELISA kit, purchased from mlbio, product number: ml106471.

[0155] Human IL-1β ELISA kit, purchased from Invitrogen, product number: 88-7261-88.

[0156] Human IL-6 ELISA kit, purchased from Beyotime, product number: PI330.

[0157] Results: The effects of compound bergamot nasal wash on inflammatory damage to nasal mucosa organoids were as follows: Figure 2 As shown, compared with the inflammation group, the expression levels of inflammatory factors in the compound bergamot nasal rinse group were significantly reduced, indicating that the addition of bergamot nasal rinse significantly inhibited the expression of various inflammatory factors in nasal mucosal organoids, including significantly inhibiting TNF-α in nasal mucosal organoids (**p<0.001). Figure 2 A) IL-1β (****p<0.00001)( Figure 2 B) IL-6 (**p<0.001) Figure 2 C) The expression of cellular inflammatory factors was reduced, and Formula 1 showed a stronger anti-inflammatory effect than Formula 2; therefore, Formula 1 is the superior formula. Example 5: Safety study of nasal mucosal organoids.

[0158] Experimental methods and efficacy indicators: The effects of compound bergamot nasal wash on nasal mucosal organoids were compared. The safety of compound bergamot nasal wash on nasal mucosal organoids was assessed by detecting ATP activity.

[0159] Subject source: Same as Example 3

[0160] Experimental method: ATP chemiluminescence method

[0161] Grouping and dosing regimen:

[0162] Experimental group 1: 100% compound nasal wash formula 1

[0163] Experimental Group 2: 100% Compound Nasal Wash Formula 2

[0164] Control group 1: 100% saline

[0165] Control group 2: 5% pH-sensitive liposome carrier, 95% physiological saline

[0166] Test kits used:

[0167] Cell Titer Glo 3D Cell Viability Assay Kit, purchased from Promega, product number: G9681.

[0168] Results: The effect of compound bergamot nasal wash on nasal mucosal organoid activity was as follows: Figure 3 As shown, overall, cell survival rate did not show a significant decreasing trend with increasing use of Compound Bergamot Nasal Wash, and the survival rate within 48 hours was not significantly different from the control group. This result is consistent with the organoid morphology observed under a light microscope. Compared with the DMSO group and the positive control group, the cells did not exhibit apoptosis morphological characteristics such as rounding and shrinkage, suggesting that Compound Bergamot Nasal Wash has high safety.

[0169] Example 6: Subject-based trial of Compound Bergamot Nasal Wash

[0170] The clinical efficacy of the compound bergamot nasal wash (formula 1) described in Example 1 in improving symptoms in patients with upper respiratory tract infections was evaluated.

[0171] Study Design: Thirty patients were randomly enrolled using a block randomization method. The study was a randomized, controlled, three-arm trial (experimental group, control group, and blank control group), and was single-blind (the investigator blinded the symptom scale assessment). Inclusion criteria were age 18-65 years, confirmed acute upper respiratory tract infection (duration ≤48 hours), and symptoms including nasal congestion, runny nose, sore throat, and cough.

[0172] The experimental group received nasal irrigation with compound bergamot nasal wash (10ml per nostril twice daily, totaling 20ml for 5 days) plus routine symptomatic treatment (acetaminophen 500-1000mg / day); the control group received nasal irrigation with saline solution (same method as the experimental group) plus routine symptomatic treatment; the blank control group received only routine symptomatic treatment and no nasal irrigation.

[0173] Evaluation indicators:

[0174] Primary endpoint: Symptom scales were completed on days 3 and 5 after diagnosis, and the sum of the total scores was used as the efficacy indicator.

[0175] Symptom scale (0-3 points for each item, 0-12 points in total): nasal congestion, runny nose, sore throat, cough.

[0176] Therapeutic effects classification:

[0177] Significant: Total score decrease of ≥70% (e.g., baseline 12 points → ≤4 points);

[0178] Effective: Total score decreases by 30%-69% (e.g., 12 points → 5-8 points);

[0179] Not obvious: Total score decreased by less than 30%.

[0180] The data statistics and results are shown in Table 5 below:

[0181] Table 5. Statistical analysis of trial results for Compound Bergamot Nasal Wash.

[0182]

[0183] The data in Table 5 show that the experimental group showed significantly better symptom improvement than the control group and the blank group, supporting the clinical value of compound bergamot nasal wash for nasal irrigation.

[0184] Comparative Example 1: Comparison of the antiviral effects of each effective monomer in Compound Bergamot Nasal Wash

[0185] The purpose of this comparative experiment was to analyze the effects of each monomeric substance in the effective components of Compound Bergamot Nasal Wash, such as bergamotin, honeysuckle extract, nano-encapsulated bamboo leaf polysaccharide, Imperata cylindrica root extract, and sodium pyruvate, on antiviral activity.

[0186] Preparation of test reagents:

[0187] Prepare nasal irrigation solutions according to the following groups:

[0188] ①: The compound bergamot nasal wash described in Example 1;

[0189] ②: The compound bergamot nasal wash described in Example 1 has removed honeysuckle extract, bamboo leaf polysaccharide encapsulated with nanomaterials, Imperata cylindrica root extract, and sodium pyruvate, leaving only bergamot extract as an active ingredient.

[0190] ③: The compound bergamot nasal wash described in Example 1 has removed bergamot, nano-encapsulated bamboo leaf polysaccharide, Imperata cylindrica root extract, and sodium pyruvate, leaving only honeysuckle extract among the active ingredients.

[0191] ④: The compound bergamot nasal wash described in Example 1 removes bergamot extract, honeysuckle extract, Imperata cylindrica root extract, and sodium pyruvate, retaining only nano-encapsulated bamboo leaf polysaccharide among the active ingredients.

[0192] ⑤: The compound bergamot nasal wash described in Example 1 has removed bergamot extract, honeysuckle extract, nano-encapsulated bamboo leaf polysaccharide, and sodium pyruvate, leaving only Imperata cylindrica root extract as the active ingredient.

[0193] ⑥: The compound bergamot nasal wash described in Example 1 has removed bergamot extract, honeysuckle extract, nano-encapsulated bamboo leaf polysaccharide, and Imperata cylindrica root extract, leaving only sodium pyruvate as an active ingredient.

[0194] All reagents in groups 1-6 were prepared according to the method described in Example 1, with each reagent prepared in 10 ml volume.

[0195] Subject Sources: Patients were collected from October 2020 to June 2023 at Nanfang Hospital, Southern Medical University, who were pathologically diagnosed with nasal polyps, bullous middle turbinate, etc., requiring surgical removal of the affected tissue. Residual tissue from fresh olfactory epithelial endoscopic surgery was also collected. All patients included in the study had signed informed consent forms (ICF) approved by the IRB / IEC before surgery. The ethics of this research project were approved by the Ethics Committee of Nanfang Hospital.

[0196] Experimental methods and efficacy indicators:

[0197] Nasal mucosal organoids were infected with SARS-CoV-2 virus (2019-nCoV-WIV04, B.1.1.7) to assess the antiviral efficacy of compound bergamot nasal wash on nasal mucosal organoids. The viral copy number in infected nasal mucosal organoids was analyzed using RT-qPCR.

[0198] Dosing regimens for the experimental and control groups:

[0199] Experimental Group 1: The total proportion of the compound nasal wash described in Group ① (bergamot extract, honeysuckle extract, bamboo leaf polysaccharide, Imperata cylindrica root extract, and sodium pyruvate) was 5.8%.

[0200] Experimental group 2: The compound nasal wash containing only 5.8% bergamot extract as described in group ②

[0201] Experimental group 3: The compound nasal wash containing only 5.8% honeysuckle extract as described in group ③

[0202] Experimental group 4: The compound nasal wash described in group ④ containing only 5.8% nano-encapsulated bamboo leaf polysaccharide.

[0203] Experimental group 5: The compound nasal wash containing only 5.8% Imperata cylindrica root extract as described in group ⑤

[0204] Experimental group 6: The compound nasal wash containing only 5.8% sodium pyruvate as described in group 6.

[0205] Control group 1: 100% saline

[0206] Result: As Figure 4As shown, the viral RNA inhibition effect of the compound bergamot nasal wash is significantly better than that of the single-component formulation. This indicates that the various components in the compound bergamot nasal wash of the present invention have achieved a synergistic effect, resulting in a significant improvement in the efficacy of the compound bergamot nasal wash.

[0207] Comparative Example 2: Comparison of anti-inflammatory effects of Compound Bergamot Nasal Wash after removing some active ingredients

[0208] This comparative study involved the intervention of LPS (1 μg / ml) nasal mucosal organoids after removing some active ingredients from compound bergamot nasal wash. The changes in the secretion levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected, and the anti-inflammatory and antioxidant effects of bergamot were assessed. This analysis compared the contributions of each component of the nasal wash to the anti-inflammatory efficacy.

[0209] Preparation of test reagents:

[0210] Prepare nasal irrigation solutions according to the following groups:

[0211] ①. The compound bergamot nasal wash described in Example 1;

[0212] ②The compound bergamot nasal wash described in Example 1 has honeysuckle extract removed.

[0213] ③The compound bergamot nasal wash described in Example 1 removes bergamotin.

[0214] ④ The compound bergamot nasal wash described in Example 1 has the Imperata cylindrica root extract removed.

[0215] ⑤ The compound bergamot nasal wash described in Example 1 removes the nano-encapsulated bamboo leaf polysaccharide.

[0216] ⑥The compound bergamot nasal wash solution described in Example 1 removes sodium pyruvate.

[0217] Subject source: Same as Example 3

[0218] Experimental method: ELISA enzyme-linked detection method

[0219] Grouping and dosing regimen:

[0220] Experimental group 1: Compound bergamot nasal wash as described in group ①

[0221] Experimental group 2: The compound bergamot nasal wash described in group ② has the honeysuckle extract removed.

[0222] Experimental group 3: The compound bergamot nasal wash described in group ③ has removed bergamot extract.

[0223] Experimental group 4: The compound bergamot nasal wash described in group ④ has the Imperata cylindrica root extract removed.

[0224] Experimental group 5: The compound bergamot nasal wash described in group ⑤ has removed the nano-encapsulated bamboo leaf polysaccharide.

[0225] Experimental group 6: The compound bergamot nasal wash described in group 6 has removed sodium pyruvate.

[0226] Control group 1: 100% saline

[0227] Control group 2: 5% pH-sensitive liposome carrier, 95% physiological saline

[0228] Test kits used:

[0229] Human TNF-α ELISA kit, purchased from mlbio, product number: ml106471

[0230] Human IL-1β ELISA kit, purchased from Invitrogen, product number: 88-7261-88

[0231] Human IL-6 ELISA kit, purchased from Beyotime, product number: PI330

[0232] Results: The effects of compound bergamot nasal wash on inflammatory damage to nasal mucosa organoids were as follows: Figure 5 As shown, compared with the inflammation group, the expression of inflammatory factors in the compound bergamot nasal wash group was significantly reduced, and the expression of inflammatory factors increased after the removal of relevant active components, proving that the active ingredients in the compound nasal wash all play a key role in reducing nasal inflammation.

[0233] Comparative Example 3: Effects of different pH levels on the anti-inflammatory efficacy of compound bergamot nasal wash

[0234] This comparative study involved the intervention of LPS (1 μg / ml) nasal mucosal organoids after removing some active ingredients from compound bergamot nasal wash. The changes in the secretion levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were detected, and the anti-inflammatory and antioxidant effects of bergamot were assessed. This analysis compared the contributions of each component of the nasal wash to the anti-inflammatory efficacy.

[0235] Preparation of test reagents:

[0236] Prepare nasal irrigation solutions according to the following groups:

[0237] ①. The compound bergamot nasal wash solution described in Example 1 has a pH adjusted to 6.5;

[0238] ②The compound bergamot nasal wash solution described in Example 1 was adjusted to pH 5.5.

[0239] ③The compound bergamot nasal wash solution described in Example 1 was adjusted to pH 7.0.

[0240] ④ The compound bergamot nasal wash described in Example 1 was adjusted to pH 8.0.

[0241] Subject source: Same as Example 3

[0242] Experimental method: ELISA enzyme-linked detection method

[0243] Grouping and dosing regimen:

[0244] Experimental group 1: The compound bergamot nasal wash described in group ①, with a pH of 6.5.

[0245] Experimental group 2: The compound bergamot nasal wash described in group ②, with a pH of 5.5.

[0246] Experimental group 3: The compound bergamot nasal wash described in group ③, with a pH of 7.0.

[0247] Experimental group 4: The compound bergamot nasal wash described in group ④, with a pH of 8.0.

[0248] Control group: 100% saline

[0249] Test kits used:

[0250] Human TNF-α ELISA kit, purchased from mlbio, product number: ml106471

[0251] Human IL-1β ELISA kit, purchased from Invitrogen, product number: 88-7261-88

[0252] Human IL-6 ELISA kit, purchased from Beyotime, product number: PI330

[0253] Results: The effects of compound bergamot nasal wash on inflammatory damage to nasal mucosa organoids were as follows: Figure 6 As shown, compound bergamot nasal wash has the best anti-inflammatory effect at pH 6.5, while lower or higher pH values ​​reduce its anti-inflammatory ability in nasal mucosal organoids.

[0254] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A compound bergamot nasal wash, characterized in that, By weight, it includes the following components: Bergamotin 1%–2%, Sodium pyruvate 0.5%–1.2%, Honeysuckle extract 1.5%–3%, Nano-encapsulated Lophatherum gracile polysaccharides 0.3%–0.8%, Imperata cylindrica root extract 0.5%–1.5%, pH-responsive liposomes: 5%–10% The remainder is physiological saline.

2. The compound bergamot nasal wash according to claim 1, characterized in that, By weight, it includes the following components: Bergamotin 1.5%, Sodium pyruvate 0.8%, Honeysuckle extract 2%, Nano-encapsulated 0.5% of bamboo leaf polysaccharide 1% Imperata cylindrica root extract pH-responsive liposomes 5%, The remainder is physiological saline.

3. The compound bergamot nasal wash according to claim 1, characterized in that, The preparation method of the nano-encapsulated bamboo leaf polysaccharide includes: mixing chitosan and bamboo leaf polysaccharide, dissolving, homogenizing, and freeze-drying to obtain the nano-encapsulated bamboo leaf polysaccharide.

4. The compound bergamot nasal wash according to claim 3, characterized in that, The mass ratio of chitosan to bamboo leaf polysaccharide is 1:(1-5).

5. The compound bergamot nasal wash according to claim 1, characterized in that, The method for preparing the pH-responsive liposomes includes: mixing DPPC, cholesterol and polyethylene glycol-polylactic acid, dissolving, evaporating the solvent to form a film, ultrasonically hydrating, and extruding to granulate, thereby obtaining the pH-responsive liposomes.

6. The compound bergamot nasal wash according to claim 5, characterized in that, The mass ratio of cholesterol to polyethylene glycol-polylactic acid in the DPPC is (5-10):(1-5):

1.

7. A method for preparing a compound bergamot nasal wash, characterized in that, Includes the following steps: By weight, 1%–2% bergamot, 0.5%–1.5% Imperata cylindrica root extract, and 5%–10% pH-responsive liposomes were dissolved in an organic solvent. After removing the solvent, a lipid membrane was obtained. Sodium pyruvate 0.5%–1.2%, honeysuckle extract 1.5%–3%, and nano-encapsulated bamboo leaf polysaccharide 0.3%–0.8% were dissolved in water to obtain a hydration medium. The hydration medium was mixed with the lipid membrane, sonicated, and extruded to obtain a drug-loaded liposome concentrate. The pH of the drug-loaded liposome concentrate was adjusted to 6.0–6.5, and the solution was filtered to remove bacteria, thus obtaining the compound bergamot nasal wash.

8. A pharmaceutical product, characterized in that, The medicine contains the compound bergamot nasal wash as described in any one of claims 1-6 and pharmaceutically permissible excipients.

9. The use of the compound bergamot nasal wash according to any one of claims 1-6 in the preparation of a medicament for treating respiratory tract infections and / or respiratory tract inflammation.

10. The application according to claim 9, characterized in that, The respiratory infections include upper respiratory tract infections.

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