A mussel adhesive protein nano-modified medicament for improving lung function and a preparation method and application thereof

Abstract

The application provides a mussel adhesion protein nano-modified medicament for improving lung function and a preparation method and application thereof, and belongs to the technical field of medicines. Lily and sophora flower bud are crushed, heated extraction is carried out through a composite enzyme extraction solution, filtration is carried out, a complex is prepared by adding ethanol to precipitate the filtrate, active substances are separated by adding immobilized adsorption materials to the filtrate, the complex, amino acids, vitamins and inorganic salts are mixed and added to water, fermentation is carried out by inoculating selenium-rich yeast, a selenium-rich glycoprotein is prepared, the selenium-rich glycoprotein, the active substances, mussel adhesion protein modified exosomes and medicinal adjuvants are uniformly mixed, filtration is carried out, filling and sealing are carried out, sterilization is carried out, and the mussel adhesion protein nano-modified medicament for improving lung function is prepared. The mussel adhesion protein nano-modified medicament for improving lung function prepared by the application has good effects of moistening the lung, resisting oxidation, resisting inflammation, resisting cancer, relieving cough, reducing phlegm, relieving asthma, protecting liver and lung functions, is helpful to respiratory health, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a mussel adhesion protein nano-modified agent for improving lung function, its preparation method, and its application. Background Technology

[0002] The development of industrial activities and the heavy energy demands of modern life have resulted in severe air pollution during energy acquisition. Harvard University research on air pollution in Asia and Southeast Asia indicates that coal-fired power plants, in addition to directly emitting PM2.5, also emit substances that indirectly promote PM2.5 formation, such as sulfur oxides (SO2), nitrogen oxides (NOx), soot, and dust. PM2.5 particles are very small, respirable particles that can easily penetrate the respiratory defense mechanisms of the nasal cavity and tracheal cilia, entering the bronchi and alveoli, causing serious health effects, including respiratory diseases, chronic bronchitis, lung cancer, and cardiovascular diseases. For pregnant women, it can lead to premature birth, impaired cognitive development, and increased incidence of chronic diseases; in severe cases, it can cause death.

[0003] However, currently, we can only mitigate or prevent the harm caused by PM2.5 through passive methods of reducing inhalation. While atmospheric PM2.5 can be reduced by decreasing outdoor activity time and wearing personal protective equipment such as masks, PM2.5 can seep into indoor environments through ventilation and air conditioning systems, doors and windows, and building gaps, increasing indoor air pollution and exposure risks. Many studies have found that indoor PM2.5 concentrations are often much higher than outdoor levels due to cooking fumes, cleaning dust, and biological sources including viruses and bacteria in droplets, mold spores, and dust mite excrement. Therefore, in addition to improving energy usage habits and developing clean energy sources, we must protect our respiratory system to avoid the harm caused by air pollution until the problem is alleviated. Furthermore, besides man-made pollution, the air also contains biological agents that threaten human health, such as bacteria and viruses that can cause respiratory infections.

[0004] In summary, in order to effectively mitigate the damage of PM2.5 to the human lungs, it is necessary to develop a composition that can effectively promote the phagocytic capacity of macrophages to increase the body's ability to eliminate PM2.5, while simultaneously effectively inhibiting the growth of bacteria in the lungs, enhancing the repair capacity of lung epithelial cells, protecting respiratory health, and prolonging retention time and improving bioavailability. This composition has broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to propose a mussel adhesion protein nano-modified agent for improving lung function, its preparation method, and its application. This invention overcomes the technical problems of existing lung-administered formulations, such as short retention time on the alveolar surface, low bioavailability, lack of specific adhesion in exosome delivery to the lungs, and low extraction efficiency and difficulty in selective separation of active ingredients from natural products such as lily and sophora japonica. It has good effects on moisturizing the lungs, anti-oxidation, anti-inflammation, and anti-cancer, as well as relieving cough, expectoration, and asthma, and protecting liver and lung function. It is beneficial to respiratory health and has broad application prospects.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing a mussel adhesion protein nano-modified pharmaceutical agent to improve lung function. Lily bulbs and Sophora japonica flowers are pulverized, extracted with a compound enzyme extract solution under heat, filtered, and the filtrate is precipitated with ethanol to obtain a complex. An immobilized adsorbent material is added to the filtrate to separate the active ingredient. The complex, amino acids, vitamins, and inorganic salts are mixed and added to water, then inoculated with selenium-enriched yeast for fermentation to obtain a selenium-enriched glycoprotein. This selenium-enriched glycoprotein is then mixed evenly with the active ingredient, mussel adhesion protein-modified exosomes, and pharmaceutical excipients, filtered, filled, and sterilized to obtain the mussel adhesion protein nano-modified pharmaceutical agent for improving lung function.

[0008] Sophora japonica buds have the effects of clearing heat and lowering blood pressure, anti-inflammation, hemostasis, expectoration and cough relief. They are suitable for treating various lung-related diseases, such as pneumonia, lung abscess, bronchitis, bronchial asthma, bronchiectasis, and lung cancer. For coughs caused by damp phlegm or cold phlegm, Sophora japonica buds can be used in combination with other traditional Chinese medicines. They have a certain auxiliary therapeutic effect on pulmonary nodules caused by acute pulmonary infectious lesions, promoting the shrinkage and disappearance of nodules. The rutin contained in Sophora japonica buds has anti-inflammatory and analgesic effects, which can reduce capillary permeability and reduce inflammatory reactions, thereby achieving anti-inflammatory and cough-relieving effects. The rutin component in Sophora japonica buds can maintain the normal resistance of capillaries and prevent bleeding symptoms. In addition, the quercetin contained in Sophora japonica buds, after fermentation, can form quercetin glycosides, which have good antioxidant and anti-inflammatory effects and can significantly improve symptoms of pneumonia, bronchitis, etc.

[0009] Lily bulbs are sweet and slightly bitter in taste, and enter the heart and lung meridians. They have the effects of moistening the lungs and relieving cough, and are suitable for treating symptoms such as dry cough with little phlegm or blood in the sputum. Lily bulbs also have the effect of nourishing yin and clearing heat, and can alleviate symptoms such as cough, dry mouth and tongue, irritability and insomnia caused by yin deficiency and internal heat. The polysaccharides and other components in lily bulbs have antioxidant effects, which can eliminate free radicals in the body and protect lung cells from oxidative damage. The combination of these two ingredients, which are both food and medicine, is widely known and has a good effect of nourishing yin and moistening the lungs.

[0010] As a further improvement to the present invention, the following steps are included:

[0011] S1. Wash, dry, and grind the lily bulbs and sophora japonica flowers to obtain a combined powder;

[0012] S2. Add the combined powder to the compound enzyme extract, heat and stir to extract, filter, inactivate the enzyme, and keep the filtrate;

[0013] S3. Add ethanol to the filtrate to precipitate, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for later use.

[0014] S4. Add immobilized adsorbent to the filtrate, treat with ultrasound to adsorb, filter, add the solid to ethanol, heat to desorb, centrifuge to separate the immobilized adsorbent, wash and recycle, remove the solvent from the liquid under reduced pressure, dry, and obtain the active product.

[0015] S5. Mix the complex, amino acids, vitamins, and inorganic salts and add them to water. Sterilize the mixture, inoculate it with selenium-enriched yeast seed solution, ferment and culture it, filter it, and dialyze the filtrate to obtain selenium-enriched glycoprotein.

[0016] S6. Mix the PBS suspension of milk exosomes with the PBS solution of mussel adhesion protein, incubate, centrifuge, and freeze-dry to obtain mussel adhesion protein modified exosomes;

[0017] S7. Selenium-enriched glycoprotein, mussel adhesive protein-modified exosomes, active ingredients, and pharmaceutical excipients are mixed evenly, filtered, filled, and sterilized to obtain a mussel adhesive protein nano-modified drug for improving lung function.

[0018] As a further improvement of the present invention, the mass ratio of lily and sophora japonica in step S1 is 3-5:10-15; the preparation method of the compound enzyme extract in step S2 is as follows: 2-3g of cellulase and 1-2g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract. The solid-liquid ratio of the combined powder and the compound enzyme extract is 1:7-10g / mL, and the heating and stirring extraction temperature is 35-40℃, and the time is 2-4h.

[0019] This invention uses a compound enzyme extract to extract the combined powder, simulating the human intestinal environment. In addition to the original digestive enzymes, cellulase and pectinase are added, which can improve the extraction rate of the effective components of traditional Chinese medicine, shorten the production cycle, reduce costs, and increase the solubility of the drug, especially for fat-soluble substances. This helps to improve the absorption rate of the drug. At the same time, the biomimetic extraction method has mild reaction conditions, saves energy, and does not pollute the environment with organic solvents, which meets the requirements of green chemistry and environmental protection.

[0020] As a further improvement of the present invention, in step S3, ethanol is added until the ethanol content of the system is 75-85 wt%, and precipitation is carried out for 3-5 hours.

[0021] As a further improvement of the present invention, the mass ratio of the filtrate to the immobilized adsorbent in step S4 is 10:1-2, the ultrasonic treatment adsorption power is 200-600W, the time is 30-40min, and the heating desorption temperature is 55-65℃; the preparation method of the immobilized adsorbent in step S4 is as follows:

[0022] T1. 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, aluminum alkoxide, Span, polyvinylpyrrolidone and ethyl acetate were dissolved in n-butanol to obtain an oil phase; Tween and pore-forming agent were dissolved in water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified, stirred, centrifuged, washed and dried to obtain nanospheres loaded with ionic liquids.

[0023] T2. Add the nanospheres loaded with ionic liquid to water, add dopamine hydrochloride and catalyst, stir the reaction, centrifuge, wash, and dry to obtain the modified nanospheres loaded with ionic liquid.

[0024] T3. Modified nanospheres loaded with ionic liquid were mixed with salt and added to acetonitrile. The mixture was stirred, filtered, washed, and dried to obtain the immobilized adsorbent material. Anion exchange was used to convert the hydrogen sulfate-type ionic liquid into a tetrafluoroborate or hexafluorophosphate type, reducing the hydrophilicity of the ionic liquid and enhancing the selective adsorption capacity of the nanospheres for flavonoid aglycones.

[0025] Ionic liquids are successfully immobilized on nanospheres via a sol-gel reaction. The surface is then modified with polydopamine, which bonds active groups such as hydroxyl, amino, and carboxyl groups to the ionic liquid structure. This facilitates interactions with target molecules through π-π bonds, hydrogen bonds, ionic bonds, and van der Waals forces, resulting in a more robust immobilization reaction and better adsorption and separation of active substances, including flavonoids, polyphenols, triterpenes, and saponins. Immobilizing ionic liquids on solid carrier materials reduces ionic liquid loss while retaining the unique properties of both the ionic liquid and the solid carrier. This method features high enrichment efficiency, high adsorption capacity, good stability, multiple recognition sites, strong extraction selectivity, and high ionic liquid utilization. Ultrasonic-assisted adsorption promotes the adsorption of active substances, while heating in a good solvent promotes desorption, thus achieving the separation and purification of active substances. This method is convenient, fast, and efficient. Furthermore, the immobilized material can be reused, reducing costs. The resulting active substances exhibit good antioxidant, anti-inflammatory, antitussive, expectorant, and liver and lung function protection effects.

[0026] As a further improvement of the present invention, in step T1, the mass ratio of 1-(4-butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, aluminum alkoxide, Span, polyvinylpyrrolidone, ethyl acetate, and n-butanol is 4-6:7-10:0.5-1:1-2:30-40:70-100, and the mass ratio of Tween, pore-forming agent, and water is 0.5-1:1-2:60-70. The Span is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85, and the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85. The pore-forming agent is hexadecyltrimethylammonium chloride or hexadecyltrimethylammonium bromide, and the aluminum alkoxide is aluminum isopropoxide or aluminum sec-butoxide. The stirring reaction time is 20-24 h. In step T2, the mass ratio of the nanospheres loaded with ionic liquid, dopamine hydrochloride, and catalyst is 12-15:3-5:0.5-1, and the catalyst is a Tris-HCl solution with pH=9-10. The stirring reaction temperature is room temperature, and the time is 3-5 h. In step T3, the mass ratio of the modified nanospheres loaded with ionic liquid and salt is 10:15-22, and the salt is NaBF4 or KPF6. The stirring reaction time is 20-24 h.

[0027] As a further improvement of the present invention, the mass ratio of the complex, amino acids, vitamins, inorganic salts and water in step S5 is 12-15:2-4:3-5:0.002-0.003:100, and the bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 The inoculum concentration is cfu / mL, the inoculum size is 1-3 v / v%, the fermentation temperature is 28-32℃, the fermentation speed is 100-200 r / min, and the fermentation time is 24-36 h. The dialysis bag used for dialysis has a molecular weight cutoff of 5-10 kDa. The amino acid is selected from at least one of methionine, valine, lysine, isoleucine, phenylalanine, threonine, leucine, and tryptophan. The vitamin is selected from at least one of vitamin C, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, folic acid, and niacin. The inorganic salt is a mixture of sodium chloride, manganese chloride, magnesium chloride, copper chloride, and ferric chloride in a mass ratio of 10:2-3:1-3:3-4:2-4.

[0028] Selenium effectively eliminates various free radicals produced in the body, inhibits the production of inflammatory mediators, promotes antibody production by lymphocytes, enhances the body's immune function and the microbial-killing activity of macrophages, and has strong antioxidant and immunomodulatory capabilities. It plays a certain role in the treatment of asthma, and selenium supplementation can help strengthen lung function and reduce the frequency of acute exacerbations of COPD. Selenium also enhances the body's immunity, increasing patients' resistance to bacteria and viruses, and plays an important role in the prevention and treatment of lung cancer. This includes controlling the cell cycle, regulating transcription factors to inhibit the synthesis of DNA, RNA, and proteins in cancer cells, thereby inhibiting tumor cell growth; regulating the anti-cancer activity of glutathione peroxidase, effectively reducing various DNA damage induced by carcinogens, protecting normal cell structure, and preventing normal cells from mutating into tumor cells; reducing the mutagenicity of carcinogens, and inhibiting tumor angiogenesis and differentiation. Simultaneously, selenium also enhances the body's anti-cancer immunity.

[0029] This invention uses the extracted complex as a carbon source, adds amino acids and other substances, and ferments it with selenium-enriched yeast to obtain a selenium-enriched glycoprotein product. On the one hand, it has good effects of moisturizing the lungs, anti-oxidation, anti-inflammation and anti-cancer, improves the immune health of the respiratory tract, improves the respiratory system, and has effects such as relieving cough and asthma.

[0030] As a further improvement of the present invention, in step S6, the protein concentration of the PBS suspension of milk exosomes is 0.5-1.5 mg / mL, the protein concentration of the PBS solution of mussel adhesion protein is 0.1-1 mg / mL, the volume ratio of the PBS suspension of milk exosomes to the PBS solution of mussel adhesion protein is 1:1-1.5, the incubation conditions are 4-10℃ for 1-2 h, the pH value of the PBS suspension of milk exosomes is 7-7.8, and the pH value of the PBS solution of mussel adhesion protein is 5-6; in step S7, the mass ratio of the selenium-enriched glycoprotein, mussel adhesion protein-modified exosomes, active ingredient, and pharmaceutical excipients is 10:3-6:1-2:120-150, the pharmaceutical excipients include an osmotic pressure regulator, a flavoring agent, and a solvent, with a mass ratio of 1-3:0.5-1:100-130, the osmotic pressure regulator is selected from at least one of sodium chloride, glucose, and sodium bicarbonate, and the solvent is purified water or physiological saline.

[0031] Existing drug delivery systems, once in the alveoli, are rapidly cleared by the mucociliary clearance system of the alveolar surface mucus layer, resulting in insufficient drug retention time at the target site and difficulty in maintaining effective concentrations, requiring frequent administration to achieve therapeutic effects. This technical solution aims to modify the surface of nanocarriers using mussel adhesion protein (MAP). By utilizing the hydrogen bonds, coordination bonds, and hydrophobic interactions formed by the 3,4-dihydroxyphenylalanine (DOPA) residues in MAP with the phospholipid bilayer and epithelial cell glycocalyx on the alveolar surface, the retention time of the drug on the alveolar surface is significantly prolonged, improving local bioavailability.

[0032] Engineered exosomes are mostly used for tumor-targeted delivery, and their surface modification strategies mainly target tumor receptors, lacking specific adhesion designs for alveolar epithelial cells or the phospholipid layer on the alveolar surface. This technical solution is the first to introduce the strong underwater adhesion mechanism of mussel adhesion proteins into an exosome lung delivery system, endowing exosomes with the ability to be fixed on the alveolar surface, filling the technological gap in the field of exosome retention modification in the lungs.

[0033] This invention further protects a mussel adhesion protein nano-modified agent for improving lung function prepared by the above-described preparation method.

[0034] This invention further protects the application of the above-mentioned mussel adhesion protein nano-modified agent for improving lung function in the preparation of drugs for the prevention and treatment of pneumonia, bronchitis, and lung cancer.

[0035] The present invention has the following beneficial effects:

[0036] 1. Significantly prolongs alveolar surface retention time, improving lung-targeted delivery efficiency.

[0037] The mussel adhesion protein (MAP) modified exosomes prepared in this invention form multiple non-covalent bonds (hydrogen bonds, coordination bonds, and hydrophobic interactions) with the phospholipid bilayer on the alveolar surface through residues. Simultaneously, the abundant 3,4-dihydroxyphenylalanine (DOPA) residues in MAP form hydrogen bonds with the phospholipid bilayer and epithelial cell glycocalyx on the alveolar surface, forming an "exosome-MAP-alveolar" sandwich adhesion structure. This structure effectively resists mucociliary clearance, significantly prolonging the retention time of exosomes on the alveolar surface compared to unmodified exosomes, and greatly improving local drug concentration and bioavailability.

[0038] 2. Multi-target synergistic intervention for lung diseases, achieving integrated repair effects.

[0039] Mussel adhesion proteins act as "bio-glue" to anchor exosomes to the alveolar surface, releasing milk exosomes. Their inherent bioactive molecules (such as miRNA and proteins) promote the regeneration and repair of alveolar epithelial cells and maintain the integrity of the alveolar-capillary barrier. Selenium-rich glycoproteins provide organic selenium, which scavenge oxygen free radicals through the glutathione peroxidase pathway, reducing oxidative stress damage. At the same time, the glycoproteins themselves have immunomodulatory activity. In the lily-sophora japonica active ingredients, the complex activates macrophages and T lymphocytes, enhancing non-specific immunity. Flavonoid active ingredients (such as rutin and quercetin) inhibit the NF-κB inflammatory pathway and reduce the levels of pro-inflammatory factors such as IL-6 and TNF-α. The above components work synergistically and are released synchronously in the alveoli, producing a synergistic therapeutic effect of "1+1>2".

[0040] 3. High extraction efficiency of active ingredients, full utilization of resources, and the immobilized adsorption material can be recycled.

[0041] The compound enzyme extract synergistically disrupts plant cell walls and cell membranes under mild conditions, significantly increasing the dissolution rate of lily polysaccharides and sophora japonica flavonoids. The supernatant after alcohol precipitation of polysaccharides is selectively adsorbed by immobilized adsorption material (polydopamine-modified ionic liquid nanospheres). The adsorption-desorption process is simple, and the immobilized material can be recycled after centrifugation and washing, reducing production costs and organic solvent consumption, which is in line with the concept of green manufacturing.

[0042] 4. Selenium-enriched glycoproteins have a well-defined organic selenium form, high safety, and good bioavailability.

[0043] Through targeted fermentation with selenium-enriched yeast, inorganic selenium is converted into organic selenium forms such as selenomethionine and selenocysteine ​​and integrated into the yeast glycoprotein backbone. Then, the product is dialyzed through a 5-10kDa dialysis bag to remove free inorganic selenium, heavy metal ions and small molecule impurities. The product has a high content of organic selenium, a clear form, and significantly better bioavailability than inorganic selenium salts, while avoiding the risk of cumulative toxicity of inorganic selenium.

[0044] 5. All-natural source, excellent biocompatibility, low immunogenicity

[0045] All components of this medicine are derived from natural substances: lily and sophora japonica are both medicinal and edible plants; milk exosomes are natural extracellular vesicles with low immunogenicity and high biocompatibility; mussel adhesive protein is a marine biological protein with good affinity for human tissue; and selenium-enriched yeast is a food-grade microorganism. While the components work synergistically, the cytotoxicity and immune rejection reactions that may be caused by chemically synthesized carriers are avoided, resulting in high safety and suitability for long-term preventative use.

[0046] 6. It has a broad range of indications and combines preventive and therapeutic value.

[0047] This invention integrates anti-inflammatory, antioxidant, immunomodulatory, and tissue repair functions. It can be used not only for inflammation control and tissue repair during the acute phase of pneumonia and bronchitis, but also as an adjuvant protective agent after radiotherapy and chemotherapy for lung cancer to reduce drug-induced lung injury. At the same time, its all-natural and low-toxicity characteristics make it suitable as a preventive lung health care preparation for high-risk groups. It has good effects on moisturizing the lungs, anti-oxidation, anti-inflammation, and anti-cancer, as well as relieving cough, expectoration, and asthma, and protecting liver and lung function. It is conducive to respiratory health and has broad application prospects. Attached Figure Description

[0048] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0049] Figure 1 The infrared spectrum of the immobilized adsorbent material prepared in Example 1.

[0050] Figure 2 The image shows the SGS-PAGE electrophoresis results of the substrate and selenium-enriched glycoprotein prepared in Example 1, where 1 is the unfermented substrate control and 2 is the selenium-enriched glycoprotein. Detailed Implementation

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0052] 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, [(n-Bu-SO3H)MIm][HSO4] ionic liquid, purchased from Lanzhou Institute of Physical Chemistry, Chinese Academy of Sciences.

[0053] Selenium-enriched yeast, 20 billion CFU / g, purchased from Angel Yeast Co., Ltd.

[0054] Cellulase, 10,000 U / g, pectinase, 25,000 U / g, purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.

[0055] Milk exosomes, purity >98%, average diameter 70-100nm.

[0056] Mussel adhesive protein, purity >90%, molecular weight 100,000, DL-DOTA content: 5.6%.

[0057] Preparation Example 1: Preparation of Immobilized Adsorbent Materials

[0058] The method is as follows:

[0059] T1. 4g of 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, 7g of aluminum isopropoxide, 0.5g of Span-40, 1g of polyvinylpyrrolidone, and 30g of ethyl acetate were dissolved in 70g of n-butanol to obtain an oil phase; 0.5g of Tween-40 and 1g of hexadecyltrimethylammonium chloride were dissolved in 60g of water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified at 7000r / min for 15min, stirred for 20h, centrifuged, washed, and dried to obtain nanospheres loaded with ionic liquid;

[0060] T2. Add 12g of nanospheres loaded with ionic liquid to 200mL of water, add 3g of dopamine hydrochloride and 0.5g of catalyst, stir at room temperature for 3h, centrifuge, wash, dry, and obtain modified nanospheres loaded with ionic liquid.

[0061] The catalyst is a Tris-HCl solution with a pH of 8.5-9.5;

[0062] T3. 10g of modified nanospheres loaded with ionic liquid were mixed with 15g of KPF6 and added to 200mL of acetonitrile. The mixture was stirred and reacted for 20h. After filtration, washing, and drying, the immobilized adsorbent material was obtained. Figure 1 The image shows the infrared spectrum of the immobilized adsorbent material. The 3400 cm⁻¹ value is shown in the image. -1 The broad peak corresponds to the -OH stretching vibration, 2920 cm⁻¹. -1 and 2850 cm -1 The position is a methylene stretching vibration, 1630 cm⁻¹ -1 The presence of polydopamine NH bending vibration at the point indicates that polydopamine was successfully coated onto the surface of nanospheres loaded with ionic liquid.

[0063] Preparation Example 2: Preparation of Immobilized Adsorbent Materials

[0064] The method is as follows:

[0065] T1. 6g of 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, 10g of aluminum sec-butoxide, 1g of Span-60, 2g of polyvinylpyrrolidone and 40g of ethyl acetate were dissolved in 100g of n-butanol to obtain an oil phase; 1g of Tween-60 and 2g of hexadecyltrimethylammonium bromide were dissolved in 70g of water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified at 7000r / min for 15min, stirred for 24h, centrifuged, washed and dried to obtain nanospheres loaded with ionic liquid;

[0066] T2. Add 15g of nanospheres loaded with ionic liquid to 200mL of water, add 5g of dopamine hydrochloride and 1g of catalyst, stir at room temperature for 5h, centrifuge, wash, dry, and obtain modified nanospheres loaded with ionic liquid.

[0067] The catalyst is a Tris-HCl solution with a pH of 8.5-9.5;

[0068] T3. 10g of modified nanospheres loaded with ionic liquid was mixed with 22g of KPF6 and added to 200mL of acetonitrile. The mixture was stirred and reacted for 24h. After filtration, washing, and drying, the immobilized adsorbent material was obtained.

[0069] Preparation Example 3: Preparation of Immobilized Adsorbent Materials

[0070] The method is as follows:

[0071] T1. 5g of 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, 8g of aluminum sec-butoxide, 0.7g of Span-85, 1.5g of polyvinylpyrrolidone, and 35g of ethyl acetate were dissolved in 85g of n-butanol to obtain an oil phase; 0.7g of Tween-85 and 1.5g of hexadecyltrimethylammonium bromide were dissolved in 65g of water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified at 7000r / min for 15min, stirred for 22h, centrifuged, washed, and dried to obtain nanospheres loaded with ionic liquid;

[0072] T2. Add 13g of nanospheres loaded with ionic liquid to 200mL of water, add 4g of dopamine hydrochloride and 0.7g of catalyst, stir at room temperature for 4h, centrifuge, wash, dry, and obtain modified nanospheres loaded with ionic liquid.

[0073] The catalyst is a Tris-HCl solution with a pH of 8.5-9.5;

[0074] T3. 10g of modified nanospheres loaded with ionic liquid was mixed with 20g of KPF6 and added to 200mL of acetonitrile. The mixture was stirred and reacted for 22h. After filtration, washing, and drying, the immobilized adsorbent material was obtained.

[0075] Comparative Preparation Example 1

[0076] The difference compared to preparation example 3 is that step T2 was not performed.

[0077] Specifically as follows:

[0078] T1. 5g of 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, 8g of aluminum sec-butoxide, 0.7g of Span-85, 1.5g of polyvinylpyrrolidone, and 35g of ethyl acetate were dissolved in 85g of n-butanol to obtain an oil phase; 0.7g of Tween-85 and 1.5g of hexadecyltrimethylammonium bromide were dissolved in 65g of water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified at 7000r / min for 15min, stirred for 22h, centrifuged, washed, and dried to obtain nanospheres loaded with ionic liquid;

[0079] T2. 10g of nanospheres loaded with ionic liquid were mixed with 20g of KPF6 and added to 200mL of acetonitrile. The mixture was stirred and reacted for 22h. After filtration, washing, and drying, the immobilized adsorbent material was obtained.

[0080] Comparative Preparation Example 2

[0081] The difference from Preparation Example 3 is that hexadecyltrimethylammonium bromide was not added in step T1.

[0082] Specifically as follows:

[0083] T1. 5g of 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, 8g of aluminum sec-butoxide, 0.7g of Span-85, 1.5g of polyvinylpyrrolidone, and 35g of ethyl acetate were dissolved in 85g of n-butanol to obtain an oil phase; 0.7g of Tween-85 was dissolved in 65g of water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified at 7000r / min for 15min, stirred for 22h, centrifuged, washed, and dried to obtain nanospheres loaded with ionic liquid;

[0084] T2. Add 13g of nanospheres loaded with ionic liquid to 200mL of water, add 4g of dopamine hydrochloride and 0.7g of catalyst, stir at room temperature for 4h, centrifuge, wash, dry, and obtain modified nanospheres loaded with ionic liquid.

[0085] The catalyst is a Tris-HCl solution with a pH of 8.5-9.5;

[0086] T3. 10g of modified nanospheres loaded with ionic liquid was mixed with 20g of KPF6 and added to 200mL of acetonitrile. The mixture was stirred and reacted for 22h. After filtration, washing, and drying, the immobilized adsorbent material was obtained. Example 1

[0087] This embodiment provides a mussel adhesion protein nano-modified agent for improving lung function, including the following steps:

[0088] S1. Wash 3g of lily bulbs, mix with 10g of sophora japonica buds, dry, and pulverize to obtain the combined powder;

[0089] S2. Add 10g of the combination powder to 70mL of the compound enzyme extract, heat to 35℃, stir and extract for 2h, filter, inactivate the enzyme, and keep the filtrate.

[0090] The preparation method of the compound enzyme extract is as follows: 2g of cellulase and 1g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract.

[0091] S3. Add ethanol to the filtrate until the ethanol content of the system is 75wt%, precipitate for 3 hours, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for use.

[0092] S4. Add 10g of the immobilized adsorbent material prepared in Example 1 to 100g of filtrate, treat with ultrasonic treatment at 600W for 30min, filter, add the solid to 100mL of ethanol, heat to 55℃ to desorb, centrifuge to separate the immobilized adsorbent material, wash and recycle, remove the solvent under reduced pressure in the liquid, dry, and obtain the active product.

[0093] S5. Mix 12g of complex, 1g of glycine, 1g of phenylalanine, 3g of vitamin C, and 2mg of inorganic salts into 100mL of water, sterilize, inoculate with selenium-enriched yeast seed solution at an inoculation amount of 1v / v%, at a temperature of 28℃ and 100r / min, ferment for 24h, physically break the cell wall, filter, and dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 5kDa for 12h to obtain selenium-enriched glycoprotein. Figure 2 SGS-PAGE electrophoresis images of the substrate and the prepared selenium-enriched glycoprotein are shown. Lane 1 is the unfermented substrate control, representing the electrophoresis image taken at 0 h after inoculation with selenium-enriched yeast. It shows the molecular weight distribution of the protein before selenium enrichment and specific glycosylation modification. Lane 2 is the selenium-enriched glycoprotein, representing the electrophoresis image taken at 28°C for 24 h after inoculation with selenium-enriched yeast. Compared with lane 1, its band distribution (e.g., more diffuse, different major bands, or molecular weight shift) indicates that the yeast performed specific glycosylation modification on the matrix protein during fermentation, resulting in an increase in the apparent molecular weight of the protein and an increase in glycan heterogeneity, which is consistent with the formation mechanism of selenium-enriched glycoprotein.

[0094] Preparation method of selenium-enriched yeast seed solution: Selenium-enriched yeast was inoculated into YPD medium (yeast extract peptone glucose medium) and activated at 30℃ and 100 r / min for 18 h to obtain a culture with a bacterial count of 10. 8 -10 9 Selenium-enriched yeast seed solution at CFU / mL.

[0095] The inorganic salt is a mixture of sodium chloride, manganese chloride, magnesium chloride, copper chloride, and ferric chloride in a mass ratio of 10:2:1:3:2.

[0096] S6. Mix 100 mL of PBS suspension of milk exosomes (pH=7.4, protein concentration of 0.5 mg / mL) with 100 mL of PBS solution of mussel adhesion protein (pH=5-6, protein concentration of 0.1 mg / mL), incubate at 4 °C for 2 h, centrifuge, freeze dry to obtain mussel adhesion protein modified exosomes;

[0097] S7. Mix 10g of selenium-enriched glycoprotein, 3g of mussel adhesive protein-modified exosomes, 1g of active ingredient, and 120g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesive protein nano-modified drug for improving lung function.

[0098] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 1:0.5:100. Example 2

[0099] This embodiment provides a mussel adhesion protein nano-modified agent for improving lung function, including the following steps:

[0100] S1. Wash 5g of lily bulbs, mix with 15g of sophora japonica buds, dry, and pulverize to obtain the combined powder;

[0101] S2. Add 10g of the combination powder to 100mL of the compound enzyme extract, heat to 40℃, stir and extract for 4h, filter, inactivate the enzyme, and keep the filtrate.

[0102] The preparation method of the compound enzyme extract is as follows: 3g of cellulase and 2g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract.

[0103] S3. Add ethanol to the filtrate until the ethanol content of the system is 85wt%, precipitate for 5h, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for use.

[0104] S4. Add 20g of the immobilized adsorbent material prepared in Example 2 to 100g of filtrate, treat with 200W ultrasonic treatment for 40min, filter, add the solid to 100mL of ethanol, heat to 65℃ to desorb, centrifuge to separate the immobilized adsorbent material, wash and recycle, remove the solvent under reduced pressure in the liquid, dry, and obtain the active product.

[0105] S5. Mix 15g of complex, 2g of alanine, 2g of leucine, 4g of vitamin C, 1g of folic acid, and 3mg of inorganic salts and add to 100mL of water. Sterilize and inoculate with selenium-enriched yeast seed solution at an inoculation amount of 3v / v%, at a temperature of 32℃ and 200r / min. Ferment and culture for 36h, physically break the cell wall, filter, and dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 10kDa for 24h to obtain selenium-enriched glycoprotein.

[0106] Preparation method of selenium-enriched yeast seed solution: Selenium-enriched yeast was inoculated into YPD medium and activated at 30℃ and 100 r / min for 24 h to obtain a culture with a bacterial count of 10. 8 -10 9 Selenium-enriched yeast seed solution at CFU / mL.

[0107] The inorganic salt is a mixture of sodium chloride, manganese chloride, magnesium chloride, copper chloride, and ferric chloride in a mass ratio of 10:3:3:4:4.

[0108] S6. Mix 100 mL of PBS suspension of milk exosomes (pH=7.4, protein concentration 1.5 mg / mL) with 150 mL of PBS solution of mussel adhesion protein (pH=5-6, protein concentration 1 mg / mL), incubate at 6 °C for 1 h, centrifuge, freeze dry to obtain mussel adhesion protein modified exosomes.

[0109] S7. Mix 10g of selenium-enriched glycoprotein, 6g of mussel adhesive protein-modified exosomes, 2g of active ingredient, and 150g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesive protein nano-modified drug for improving lung function.

[0110] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 3:1:130. Example 3

[0111] This embodiment provides a mussel adhesion protein nano-modified agent for improving lung function, including the following steps:

[0112] S1. Wash 4g of lily bulbs, mix with 12g of sophora japonica buds, dry, and pulverize to obtain the combined powder;

[0113] S2. Add 10g of the combination powder to 85mL of the compound enzyme extract, heat to 37℃, stir and extract for 3h, filter, inactivate the enzyme, and keep the filtrate.

[0114] The preparation method of the compound enzyme extract is as follows: 2.5g of cellulase and 1.5g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract.

[0115] S3. Add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 4 hours, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for use.

[0116] S4. Add 15g of the immobilized adsorbent material prepared in Example 3 to 100g of filtrate, treat with ultrasonic treatment at 400W for 35min, filter, add the solid to 100mL of ethanol, heat to 60℃ to desorb, centrifuge to separate the immobilized adsorbent material, wash and recycle, remove the solvent under reduced pressure in the liquid, dry, and obtain the active product.

[0117] S5. Mix 13g of complex, 1g of glycine, 1g of phenylalanine, 1g of threonine, 2g of vitamin C, 1g of vitamin A, 1g of folic acid, and 2.5mg of inorganic salts and add to 100mL of water. Sterilize and inoculate with selenium-enriched yeast seed solution at an inoculation amount of 2v / v%, at a temperature of 30℃ and a speed of 150r / min. Ferment and culture for 30h, physically break the cell walls, filter, and dialyze the filtrate through a dialysis bag with a molecular weight cutoff of 7kDa for 18h to obtain selenium-enriched glycoprotein.

[0118] Preparation method of selenium-enriched yeast seed solution: Selenium-enriched yeast was inoculated into YPD medium and activated at 30℃ and 100 r / min for 22 h to obtain a culture with a bacterial count of 10. 8 -10 9 Selenium-enriched yeast seed solution at CFU / mL.

[0119] The inorganic salt is a mixture of sodium chloride, manganese chloride, magnesium chloride, copper chloride, and ferric chloride in a mass ratio of 10:2.5:2:3.5:3.

[0120] S6. Mix 100 mL of PBS suspension of milk exosomes (pH=7.4, protein concentration of 1 mg / mL) with 120 mL of PBS solution of mussel adhesion protein (pH=5-6, protein concentration of 0.5 mg / mL), incubate at 5 °C for 1.5 h, centrifuge, freeze dry to obtain mussel adhesion protein modified exosomes.

[0121] S7. Mix 10g of selenium-enriched glycoprotein, 4.5g of mussel adhesive protein-modified exosomes, 1.5g of active ingredient, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesive protein nano-modified drug for improving lung function.

[0122] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0123] Comparative Example 1

[0124] The difference from Example 3 is that lilies were not added in step S1.

[0125] Specifically as follows:

[0126] S1. Use 16g of Sophora japonica flowers as powder.

[0127] Comparative Example 2

[0128] The difference from Example 3 is that Sophora japonica flowers were not added in step S1.

[0129] Specifically as follows:

[0130] S1. Wash 16g of lily bulbs, dry them, and crush them to obtain the combined powder.

[0131] Comparative Example 3

[0132] The difference from Example 3 is that the complex enzyme extract in step S2 is replaced by water heating and boiling extraction.

[0133] Specifically as follows:

[0134] S2. Add 10g of the combined powder to 85mL of water, heat to boiling and stir to extract for 3 hours, filter, inactivate enzymes, and keep the filtrate.

[0135] Comparative Example 4

[0136] The difference from Example 3 is that the immobilized adsorbent material in step S4 was prepared by Comparative Preparation Example 1.

[0137] Comparative Example 5

[0138] The difference from Example 3 is that the immobilized adsorbent material in step S4 was prepared from Comparative Preparation Example 2.

[0139] Comparative Example 6

[0140] The difference from Example 3 is that step S5 was not performed.

[0141] Specifically as follows:

[0142] S1. Wash 4g of lily bulbs, mix with 12g of sophora japonica buds, dry, and pulverize to obtain the combined powder;

[0143] S2. Add 10g of the combination powder to 85mL of the compound enzyme extract, heat to 37℃, stir and extract for 3h, filter, inactivate the enzyme, and keep the filtrate.

[0144] The preparation method of the compound enzyme extract is as follows: 2.5g of cellulase and 1.5g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract.

[0145] S3. Add ethanol to the filtrate until the ethanol content of the system is 80wt%, precipitate for 4 hours, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for use.

[0146] S4. Add 15g of the immobilized adsorbent material prepared in Example 3 to 100g of filtrate, treat with ultrasonic treatment at 400W for 35min, filter, add the solid to 100mL of ethanol, heat to 60℃ to desorb, centrifuge to separate the immobilized adsorbent material, wash and recycle, remove the solvent under reduced pressure in the liquid, dry, and obtain the active product.

[0147] S5. Mix 100 mL of PBS suspension of milk exosomes (pH=7.4, protein concentration of 1 mg / mL) with 120 mL of PBS solution of mussel adhesion protein (pH=5-6, protein concentration of 0.5 mg / mL), incubate at 5 °C for 1.5 h, centrifuge, freeze dry to obtain mussel adhesion protein modified exosomes.

[0148] S6. Mix 10g of the complex, 4.5g of mussel adhesion protein-modified exosomes, 1.5g of active ingredient, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesion protein nano-modified drug for improving lung function.

[0149] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0150] Comparative Example 7

[0151] The difference from Example 3 is that no active ingredient was added in step S7.

[0152] Specifically as follows:

[0153] S7. Mix 10g of selenium-enriched glycoprotein, 4.5g of mussel adhesive protein-modified exosomes, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesive protein nano-modified drug for improving lung function.

[0154] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0155] Comparative Example 8

[0156] The difference from Example 3 is that selenium-enriched glycoprotein was not added in step S7.

[0157] Specifically as follows:

[0158] S7. Mix 4.5g of mussel adhesion protein-modified exosomes, 1.5g of active ingredient, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesion protein nano-modified drug for improving lung function.

[0159] The pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0160] Comparative Example 9

[0161] The difference from Example 3 is that in step S7, the mussel adhesion protein modified exosomes are replaced by an equal mass of milk exosomes.

[0162] S7. Mix 10g of selenium-enriched glycoprotein, 4.5g of milk exosomes, 1.5g of active ingredient, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesion protein nano-modified agent for improving lung function; the pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0163] Comparative Example 10

[0164] The difference from Example 3 is that mussel adhesion protein was not added to modify the exosomes in step S7.

[0165] S7. Mix 10g of selenium-enriched glycoprotein, 1.5g of active ingredient, and 135g of pharmaceutical excipients evenly, filter, fill, and sterilize to obtain a mussel adhesion protein nano-modified agent for improving lung function; the pharmaceutical excipients include sodium bicarbonate, lemon flavoring, and physiological saline in a mass ratio of 2:0.7:110.

[0166] Test Example 1

[0167] The active ingredients prepared in Examples 1-3 or Comparative Examples 1-5 of the present invention were subjected to antioxidant capacity tests, and the active ingredients were formulated to a concentration of 0.01 mg / mL.

[0168] Transfer 1.0 mL of the sample solution to a 10 mL stoppered colorimetric tube, add 1.0 mL each of 0.006 mol / L ferrous sulfate solution and 0.006 mol / L H₂O₂ solution, shake well, and let stand for 10 min. Add 1.0 mL of 0.006 mol / L salicylic acid solution, incubate at 37℃ for 30 min, and measure the absorbance A0 at 510 nm. Then measure the absorbance A1 of the control group (using an equal volume of distilled water instead of H₂O₂ solution) and the absorbance A2 of the blank group (replacing 1.0 mL of polyphenol solution with 1.0 mL of ultrapure water in the experimental group). Use a VC solution of equal concentration as a control and measure the scavenging rate under the same conditions. The results are shown in Table 1. The calculation formula is as follows:

[0169] ·OH radical scavenging rate (%) = 1 - (A0 - A1)A2 × 100

[0170] Table 1

[0171] As can be seen from the table above, the active ingredients extracted in Examples 1-3 of this invention have good antioxidant activity.

[0172] Test Example 2

[0173] Male C57BL / 6 mice were acclimatized for 7 days and then randomly divided into four groups: a blank control group, a model group, Example 1-3 groups, Comparative Examples 1-10 groups, and a dexamethasone group, with 10 mice in each group. Example 1-3 and Comparative Examples 1-10 groups received prophylactic administration of 1 g / day per mouse via gavage for one week. The blank control group and model group received an equal volume of physiological saline. The dexamethasone group received a prophylactic administration of 5 mg / kg per mouse via gavage once. An acute lung injury model was established by intratracheal infusion of 10 mg / kg lipopolysaccharide solution in the model group, Example 1-3 groups, Comparative Examples 1-8 groups, and the dexamethasone group. Simultaneously, the blank control group received an equal volume of physiological saline via intratracheal infusion.

[0174] Intratracheal instillation method: Mice were anesthetized with sodium amobarbital (10 mg / kg), their limbs were fixed to a foam board, and their necks were cut open to expose the trachea. A 1 mL syringe was inserted into the trachea of ​​the mouse through the tracheal wall, and 50 μL of lipopolysaccharide solution was rapidly instilled into the trachea. After completion, the foam board was shaken in four directions (up, down, left, and right) to ensure that the lipopolysaccharide solution was evenly distributed in each lung lobe.

[0175] Lung tissue pathological evaluation: Mice were anesthetized and sacrificed 24 h after stimulation with lipopolysaccharide solution. The left lung tissue of the mice was then taken, preserved in 4% paraformaldehyde fixative, fixed at room temperature for 48 h, embedded in paraffin, and sectioned to 4 μm in a microtome before fixation. Hematoxylin and eosin (HE) staining was performed, and the pathological changes of the lung tissue were observed under a light microscope. Five fields of view were randomly selected from each section, and the lung injury was scored from 0 to 4 points according to edema, necrosis, alveolar and interstitial inflammation, and hemorrhage: 0, no injury; 1, 25% injury; 2, 50% injury; 3, 75% injury; 4, 100% injury.

[0176] Myeloperoxidase (MPO) activity assay: Mice were anesthetized and euthanized after 24 h of stimulation with lipopolysaccharide solution. The upper lobe of the right lung was harvested, tissue homogenate was prepared, and MPO activity was measured using a myeloperoxidase activity assay kit.

[0177] The results are shown in Table 2.

[0178] Table 2

[0179] Note: * indicates P<0.05 compared with the blank control group; # indicates P<0.05 compared with the model group.

[0180] As shown in the table above, the mussel adhesion protein nano-modified agents for improving lung function prepared in Examples 1-3 of this invention have good protective effects against lung damage and reduce MPO activity.

[0181] Mice were euthanized under anesthesia, and blood was collected from their eyes. The levels of TNF-α and IL-6 in mouse serum were detected using an ELISA kit.

[0182] The results are shown in Table 3.

[0183] Table 3

[0184] Note: * indicates P<0.05 compared with the blank control group; # indicates P<0.05 compared with the model group.

[0185] As shown in the table above, the mussel adhesion protein nano-modified agents for improving lung function prepared in Examples 1-3 of this invention have good anti-inflammatory effects.

[0186] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a mussel adhesion protein nano-modified agent to improve lung function, characterized in that, Lily bulbs and Sophora japonica flowers were pulverized, extracted with a compound enzyme extract solution by heating, filtered, and the filtrate was precipitated with ethanol to obtain a complex. Immobilized adsorption material was added to the filtrate to separate the active ingredient. The complex, amino acids, vitamins, and inorganic salts were mixed and added to water, and then inoculated with selenium-enriched yeast for fermentation to obtain a selenium-enriched glycoprotein. This selenium-enriched glycoprotein was then mixed with the active ingredient, mussel adhesion protein-modified exosomes, and pharmaceutical excipients, filtered, filled, and sterilized to obtain a mussel adhesion protein nano-modified drug for improving lung function.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Wash, dry, and grind the lily bulbs and sophora japonica flowers to obtain a combined powder; S2. Add the combined powder to the compound enzyme extract, heat and stir to extract, filter, inactivate the enzyme, and keep the filtrate; S3. Add ethanol to the filtrate to precipitate, filter, wash the solid, dry, and obtain the complex. Recover the ethanol from the filtrate and keep it for later use. S4. Add immobilized adsorbent to the filtrate, treat with ultrasound to adsorb, filter, add the solid to ethanol, heat to desorb, centrifuge to separate the immobilized adsorbent, wash and recycle, remove the solvent from the liquid under reduced pressure, dry, and obtain the active product. S5. Mix the complex, amino acids, vitamins, and inorganic salts and add them to water. Sterilize the mixture, inoculate it with selenium-enriched yeast seed solution, ferment and culture it, filter it, and dialyze the filtrate to obtain selenium-enriched glycoprotein. S6. Mix the PBS suspension of milk exosomes with the PBS solution of mussel adhesion protein, incubate, centrifuge, and freeze-dry to obtain mussel adhesion protein modified exosomes; S7. Selenium-enriched glycoprotein, mussel adhesive protein-modified exosomes, active ingredients, and pharmaceutical excipients are mixed evenly, filtered, filled, and sterilized to obtain a mussel adhesive protein nano-modified drug for improving lung function.

3. The preparation method according to claim 2, characterized in that, The mass ratio of lily and sophora japonica buds in step S1 is 3-5:10-15; the preparation method of the compound enzyme extract in step S2 is as follows: 2-3g of cellulase and 1-2g of pectinase are added to 300mL of disodium hydrogen phosphate-citric acid buffer solution with pH=4.5-6, and stirred and mixed evenly to obtain the compound enzyme extract. The solid-liquid ratio of the combined powder and the compound enzyme extract is 1:7-10g / mL. The heating and stirring extraction temperature is 35-40℃, and the time is 2-4h.

4. The preparation method according to claim 2, characterized in that, In step S3, ethanol is added until the ethanol content in the system is 75-85 wt%, and precipitation is carried out for 3-5 hours.

5. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of the filtrate to the immobilized adsorbent is 10:1-2; the ultrasonic treatment adsorption power is 200-600W; the time is 30-40 min; and the heating desorption temperature is 55-65℃. The preparation method of the immobilized adsorbent is as follows: T1. 1-(4-Butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, aluminum alkoxide, Span, polyvinylpyrrolidone and ethyl acetate were dissolved in n-butanol to obtain an oil phase; Tween and pore-forming agent were dissolved in water to obtain an aqueous phase; the aqueous phase was added to the oil phase, emulsified, stirred, centrifuged, washed and dried to obtain nanospheres loaded with ionic liquids. T2. Add the nanospheres loaded with ionic liquid to water, add dopamine hydrochloride and catalyst, stir the reaction, centrifuge, wash, and dry to obtain the modified nanospheres loaded with ionic liquid. T3. Modified nanospheres loaded with ionic liquid were mixed with salt and added to acetonitrile. The mixture was stirred and reacted, then filtered, washed, and dried to obtain the immobilized adsorbent material.

6. The preparation method according to claim 5, characterized in that, In step T1, the mass ratio of 1-(4-butylsulfonic acid)-3-methylimidazolium hydrogen sulfate, aluminum alkoxide, Span, polyvinylpyrrolidone, ethyl acetate, and n-butanol is 4-6:7-10:0.5-1:1-2:30-40:70-100; the mass ratio of Tween, pore-forming agent, and water is 0.5-1:1-2:60-70; the Span is selected from at least one of Span-20, Span-40, Span-60, Span-80, and Span-85; the Tween is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80, and Tween-85; and the pore-forming agent is hexadecyltrimethylammonium chloride. The reaction mixture consists of methylammonium chloride or hexadecyltrimethylammonium bromide, wherein the aluminum alkoxide is aluminum isopropoxide or aluminum sec-butoxide, and the stirring reaction time is 20-24 h; in step T2, the mass ratio of the nanospheres loaded with ionic liquid, dopamine hydrochloride, and catalyst is 12-15:3-5:0.5-1, wherein the catalyst is a Tris-HCl solution with pH=8.5-9.5, and the stirring reaction temperature is room temperature for 3-5 h; in step T3, the mass ratio of the modified nanospheres loaded with ionic liquid and salt is 10:15-22, wherein the salt is NaBF4 or KPF6, and the stirring reaction time is 20-24 h.

7. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of the complex, amino acids, vitamins, inorganic salts, and water is 12-15:2-4:3-5:0.002-0.003:100, and the bacterial count of the selenium-enriched yeast seed solution is 10. 8 -10 9 The inoculum concentration is cfu / mL, the inoculum size is 1-3 v / v%, the fermentation temperature is 28-32℃, the fermentation speed is 100-200 r / min, and the fermentation time is 24-36 h. The dialysis bag used for dialysis has a molecular weight cutoff of 5-10 kDa. The amino acid is selected from at least one of methionine, valine, lysine, isoleucine, phenylalanine, threonine, leucine, and tryptophan. The vitamin is selected from at least one of vitamin C, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, folic acid, and niacin. The inorganic salt is a mixture of sodium chloride, manganese chloride, magnesium chloride, copper chloride, and ferric chloride in a mass ratio of 10:2-3:1-3:3-4:2-4.

8. The preparation method according to claim 2, characterized in that, In step S6, the protein concentration of the PBS suspension of milk exosomes is 0.5-1.5 mg / mL, the protein concentration of the PBS solution of mussel adhesion protein is 0.1-1 mg / mL, the volume ratio of the PBS suspension of milk exosomes to the PBS solution of mussel adhesion protein is 1:1-1.5, the incubation conditions are 4-10℃ for 1-2 hours, the pH value of the PBS suspension of milk exosomes is 7-7.8, and the pH value of the PBS solution of mussel adhesion protein is 5-6. In step S7, the mass ratio of the selenium-enriched glycoprotein, mussel adhesion protein-modified exosomes, active ingredient, and pharmaceutical excipients is 10:3-6:1-2:120-150, the pharmaceutical excipients include an osmotic pressure regulator, a flavoring agent, and a solvent, with a mass ratio of 1-3:0.5-1:100-130, the osmotic pressure regulator is at least one of sodium chloride, glucose, and sodium bicarbonate, and the solvent is purified water or physiological saline.

9. A mussel adhesion protein nano-modified agent for improving lung function, prepared by the method according to any one of claims 1-8.

10. The use of a mussel adhesion protein nano-modified agent for improving lung function as described in claim 9 in the preparation of drugs for the prevention and treatment of pneumonia, bronchitis, and lung cancer.

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