An anthocyanin nano-sustained release delivery system for oral lung targeting, its preparation method and application

Abstract

The present invention belongs to the technical field of food nutrition, and particularly relates to an anthocyanin nano-sustained release delivery system for oral lung targeting, its preparation method and application. Casein powder is dissolved in NaOH solution, the pH is adjusted, an acylation reagent is added, the reaction is terminated by adding NaOH solution, and after dialysis, it is freeze-dried to obtain acylated casein; the acylated casein is dissolved in water, an anthocyanin dilute HCl solution is added, and NaOH solution is added to neutralize HCl; an acylated casein-anthocyanin complex is obtained; phospholipid powder is added to ethanol and ultrasonicated until completely dissolved, and then added to the acylated casein-anthocyanin complex solution, stirred, and placed under the action of a magnetic field to self-assemble into a "molecular cap" structure. The system solution is taken out and left standing at room temperature to obtain an anthocyanin nano-sustained release delivery system. Compared with the existing lung targeting technology, the materials used in the present invention are all food-grade, safe and effective, and oral lung enrichment can be achieved without complex modification of the targeting trigger agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of food nutrition, and particularly relates to an anthocyanin nano-sustained release delivery system for oral lung targeting, a preparation method thereof, and an application thereof. Background Art

[0002] "The lungs age first as people get old". The lungs are an important respiratory organ of the human body and are also one of the most vulnerable organs. The respiratory tract is directly in contact with the outside world and is easily damaged due to long exposure time and inability to fully recover after damage. Research shows that some pneumonia patients also have sequelae after recovery, such as general weakness, shortness of breath leading to limited physical function, and the lungs showing restrictive ventilation disorders. In individual severe patients, pulmonary fibrosis may occur. At the same time, according to the latest "Annual Report on Chronic Obstructive Pulmonary Disease of the National Center for Respiratory Medicine", air pollution is related to the decline of adult lung function and will increase the risk of hospitalization for chronic obstructive pulmonary disease. In addition, the lungs of smokers are easily damaged. As of 2022, the number of smokers in China exceeded 316 million, accounting for 1 / 4 of the total population. The above data all indicate that lung health has become a major issue faced by today's society. However, lung health management cannot rely solely on drug treatment during illness, and attention should also be paid to lung health in daily life. Therefore, there is an urgent need to develop a precise nutritional food that can protect the lungs in daily life.

[0003] Research and transformation around "precision nutrition" have now become a hot topic in food and nutrition science at home and abroad. The latest evaluation by the National Institutes of Health in the United States believes that precision nutrition answers the questions of "how to eat and how to stay healthy". It is unanimously recognized by domestic and foreign scholars that with the continuous improvement of people's health awareness, public nutrition targeting "groups" can no longer meet the needs, and "precision nutrition" focusing on the "individual" health status will become a new health trend and research hotspot. Nowadays, people are no longer satisfied with just eating enough, but have shifted their goal from "eating enough" to "eating right". Therefore, precision nutritional food is a new development direction for national health.

[0004] Anthocyanins are a class of natural pigments with various biological activities produced by the combination of anthocyanidins and sugar units. They are widely distributed in the leaves, flowers, fruits and seeds of plants in nature, imparting red, purple and blue colors to nature. Anthocyanins exhibit a variety of physiological and health functions due to their strong ability to scavenge free radicals in the body, and are of great significance for dietary nutrition and the prevention of chronic diseases. The Chinese Nutrition Society put forward the specific recommended values of 6 types of phytochemicals (intake levels for healthy adults to prevent non-communicable diseases) in the "Dietary Reference Intakes for Chinese Residents (2013 Edition)", and the specific recommended value of anthocyanins is 50 mg / d. Research shows that anthocyanins have obvious protective and intervening effects on the lungs, and have good therapeutic effects on acute / chronic lung injury, pulmonary fibrosis, damage caused by air pollutants and pneumonia. However, limited by the short half-life of anthocyanins themselves, about 0.5 h, and the low blood drug concentration in the lungs, only 0 - 5 nmol, anthocyanins cannot effectively achieve oral lung intervention. At present, there is no targeted application method for the health care and treatment research of anthocyanins on the lungs, which greatly limits the physiological activity effects of anthocyanins. As an important nutritional factor in food, if anthocyanins can achieve oral targeted enrichment in the lungs, it will be of great significance for lung health care and precision nutrition.

[0005] Modern nutrition has entered the era of precision and individualization, and targeted nutrition and targeted therapy have also received unprecedented attention. Whether it is passive targeting, active targeting or physicochemical targeting, the improvement of their therapeutic effects is mostly achieved through intravascular administration. Although intravascular administration can ensure the bioavailability of nutrients or therapeutic agents to the greatest extent, it is very unfriendly to people who need long-term injection-based intravascular administration. Therefore, how to achieve the conversion of targeted drug delivery from injection to oral administration has always been a hot topic and a difficult point! Therefore, the present invention prepared an acylated casein-phospholipid-anthocyanin nano-system, which achieved the oral lung-targeted enrichment effect of anthocyanins through the regulation of charge and nano-scale, and C max is 4.7 times that of normal anthocyanins, and AUC (0-t) is 25.7 times that of normal anthocyanins, and T 1 / 2 was extended from 0.321 h to 1.174 h. Summary of the Invention

[0006] The purpose of the present invention is to construct an anthocyanin nano-system to achieve oral lung-targeted enrichment of loaded anthocyanin molecules and produce a sustained release effect in the lungs for 12 h. Another purpose of the present invention is to provide ideas and methods for anthocyanin precision nutrition.

[0007] An anthocyanin nano-sustained release delivery system for oral lung targeting, and the preparation method includes the following steps:

[0008] Step 1: Preparation of acylated casein. Dissolve casein powder in NaOH solution, adjust the pH to 7, add an acylation reagent, add NaOH solution to terminate the reaction, and after dialysis, freeze-dry to obtain acylated casein for standby.

[0009] Step 2: Preparation of acylated casein - anthocyanin complex. Dissolve acylated casein in water, and after complete dissolution, add dilute HCl solution of anthocyanin, and add NaOH solution to neutralize the HCl introduced by the anthocyanin solution; obtain acylated casein - anthocyanin complex for standby.

[0010] Step 3: Preparation of anthocyanin nano - sustained release delivery system. Add phospholipid powder to ethanol and ultrasonicate until completely dissolved, then add it to the acylated casein - anthocyanin complex solution, stir, place it under the action of a magnetic field, and self - assemble to form a "molecular cap" - like structure. Take out the system solution and let it stand at room temperature to obtain the anthocyanin nano - sustained release delivery system.

[0011] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 1, the acylation reagent is acetic acid or propionic acid or butyric acid, and the molar ratio of the acylation reagent to casein is 3:1 - 5:1.

[0012] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 1, the concentration of the NaOH solution for dissolving casein is 0.05 mol / L, the concentration of the NaOH solution for terminating the reaction is 0.05 mol / L, and the addition volume is stopped when the pH = 7.

[0013] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 2, the concentration of the dilute HCl solution is 0.01 - 0.02 mol / L.

[0014] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 2, the molar ratio of anthocyanin to acylated casein is 1:4 - 1:7.

[0015] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 3, the concentration of the phospholipid ethanol solution is 15 - 25 mg / mL.

[0016] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 3, the mass ratio of phospholipid to acylated casein is 1:1 - 1:2.

[0017] For the above - mentioned anthocyanin nano - sustained release delivery system for oral lung targeting, in Step 3, the stirring is uniform magnetic stirring in a 40 °C water bath for 1 h to fully mix the two.

[0018] For the above-mentioned anthocyanin nano-sustained release delivery system for oral lung targeting, in step three, the magnetic field strength is controlled between 1-5 mT, the magnetization time should be controlled between 5-10 min, and the magnetization temperature should be controlled between 25-45 °C.

[0019] Application of the above-mentioned anthocyanin nano-sustained release delivery system for oral lung targeting in the preparation of drugs for treating lung injury.

[0020] The application form can be in powder and solution states. For solid-state applications or applications after liquid dissolution, the maximum temperature should be controlled not to exceed 80 °C.

[0021] The content and volume involved in the present invention can be scaled up proportionally and the same preparation effect can be obtained.

[0022] Note: The main raw materials used in the preparation process of nano-systems such as acetic acid (GB 1886.10-2015), propionic acid (GB 1886.210-2016), butyric acid (GB 1886.121-2015), casein (GB 31638-2016), and phospholipids (GB 1886.358-2022) all meet the requirements of the "National Food Safety Standard". Other auxiliary chemical reagents are all food-grade and can be removed after preparation.

[0023] Oral targeting technology has characteristics such as good compliance and convenient administration, and is an effective and precise nutrition development direction to replace intravascular administration. The main raw materials used in the present invention are all food-grade. After changing the isoelectric point of casein through acylation, it is complexed with anthocyanins to form an acyl-casein-anthocyanin system, and phospholipids are further coated on the outer layer. During the mixing preparation process, low-frequency magnetic field intervention is used to make the viscous phospholipids distribute on the surface layer, and finally a self-assembled anthocyanin nano-system with a "molecular cap" effect is formed. Based on the above preparation theoretical basis, the present invention explores different preparation conditions and processes (mainly considering: ratio, preparation solvent, preparation time, magnetic field strength and time for investigation and optimization), and finally realizes the preparation of the anthocyanin nano-system. The prepared nano-system has a significant "oral" lung targeting effect and has a 12-hour sustained release effect. This anthocyanin lung targeting nano-system has a better effect on improving acute / chronic lung injury in the lungs compared with the anthocyanin prototype and has excellent potential for precise nutrition application in the lungs.

[0024] The present invention has the following beneficial effects:

[0025] ① For the first time, the "oral" targeting function of anthocyanin nutritional components is realized. The intake method has good compliance and clear efficacy, providing an effective reference for precise nutrition in conventional diets.

[0026] ② The anthocyanin nanosystem prepared by the present invention has a significant oral lung-targeted enrichment effect, and at the same time, the nanosystem shows a long-acting lung sustained-release effect for 12 h.

[0027] ③ The anthocyanin lung-targeted nanosystem prepared by the present invention has a better effect on improving acute / chronic lung injury compared with the anthocyanin prototype.

[0028]

[0029] ④ The raw materials used for preparing the anthocyanin nanosystem of the present invention are all food-grade, and the preparation method is simple, efficient, and has a high repetition rate.

[0030] It has the characteristics of scale-up production and has strong application value. Brief Description of the Drawings

[0031] Figure 1 are TEM pictures of the anthocyanin nanosystem: TEM picture stained with phosphotungstic acid (A), unstained TEM picture (B).

[0032] Figure 2 is the particle size distribution diagram in the anthocyanin nanoparticle solution.

[0033] Figure 3 are CLSM images of mouse organ tissues after oral administration of Cy5.5-labeled CLS nanoparticles.

[0034] Figure 4 are the concentrations of C3G measured by LC-MS / MS in organ tissues at different times: anthocyanin content in plasma (A), anthocyanin content in lung tissue (B), anthocyanin content in spleen tissue (C), anthocyanin content in heart tissue (D), anthocyanin content in liver tissue (E), anthocyanin content in kidney tissue (F).

[0035] Figure 5 are the tissue morphologies of C57BL / 6J mice with acute lung injury after different intervention treatments: (A) lung tissue diagram, (B) HE staining diagram of lung tissue, (C) Masson staining diagram of lung tissue.

[0036] Figure 6 are the tissue morphologies of C57BL / 6J mice with acute lung injury after different intervention treatments: (A) Tunel staining diagram of lung tissue, (B) Nrf2 immunohistochemistry diagram of lung tissue, (C) ROS change in lung tissue.

[0037] Figure 7 are the changes in physiological indexes of C57BL / 6J mice with acute lung injury after different intervention treatments: (A) lung index, (B) wet / dry weight ratio of lung, (C) Nrf2 expression level in lung tissue, (D) apoptosis rate of lung tissue cells, (E) ROS content in lung tissue.

[0038] Figure 8 Changes in physiological indexes of mice with acute lung injury after different interventions: (A) TNF-α content in lung tissue, (B) IL-1β content in lung tissue, (C) IL-6 content in lung tissue, (D) IL-10 content in lung tissue, (E) iNOS content in lung tissue, (F) COX-2 content in lung tissue, (G) CAT content in lung tissue, (H) SOD content in lung tissue, (I) GSH content in lung tissue, (J) MDA content in lung tissue.

[0039] Figure 9 Changes in tissue morphology and physiological indexes of SD rats with chronic lung injury after different interventions: (A) Overall lung morphology, (B) HE staining map of lung tissue, (C) Masson staining map of lung tissue, (D) Nrf2 expression map of lung tissue, (E) Lung index, (F) Lung wet / dry weight ratio, (G) Nrf2 expression in lung tissue.

[0040] Figure 10 Changes in tissue morphology and physiological indexes of SD rats with chronic lung injury after different interventions: (A) Tunel staining map of lung tissue: A1 represents PI staining, A2 represents DAPI staining, A3 represents the superimposed map of PI + DAPI staining, (B) ROS content in lung tissue, (C) Change in apoptosis rate of lung tissue, (D) Change in ROS content in lung tissue.

[0041] Figure 11 Changes in physiological indexes of SD rats with chronic lung injury after different interventions: (A) TNF-α content in lung tissue, (B) IL-1β content in lung tissue, (C) IL-6 content in lung tissue, (D) IL-10 content in lung tissue, (E) iNOS content in lung tissue, (F) COX-2 content in lung tissue, (G) CAT content in lung tissue, (H) SOD content in lung tissue, (I) GSH content in lung tissue, (J) MDA content in lung tissue. Specific implementation mode

[0042] Example 1 Preparation of anthocyanin nano-sustained release delivery system

[0043] Dissolve the casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to around 7 with PBS, homogenize it at 60 °C for 1 h. Add the acylating reagent acetic acid with a molar ratio of acetic acid to casein of 3:1. Dropwise add it at a flow rate of 5 ml / min and stir magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH = 7. After dialysis of the reaction solution, freeze-dry it. Take a certain amount of acylated casein and ultrasonically assist in dissolving it in an aqueous solution. Add 0.01 mol / L HCl solution of anthocyanin. The molar ratio of anthocyanin to acylated casein is 1:4. Dropwise add it while stirring, and dropwise add it at a flow rate of 5 ml / min in a 40 °C water bath and stir magnetically and evenly for 30 min. Then add an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then add the phospholipid powder to ethanol to prepare a 15 mg / mL phospholipid ethanol solution. Ultrasonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution. The mass ratio of phospholipid to acylated casein added is 1:1. Stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then place it under the action of a magnetic field with a magnetic field strength of 2 mT, a time of 5 min, and a temperature of 25 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nano-sustained release delivery system (such as Figure 1 ).

[0044] Preparation of Anthocyanin Nano-Sustained Release Delivery System in Example 2

[0045] Dissolve the casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to around 7 with PBS, homogenize it at 60 °C for 1 h. Add the acylating reagent acetic acid with a molar ratio of acetic acid to casein of 5:1. Dropwise add it at a flow rate of 5 ml / min and stir magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH = 7. After dialysis of the reaction solution, freeze-dry it. Take a certain amount of acylated casein and ultrasonically assist in dissolving it in an aqueous solution. Add 0.02 mol / L HCl solution of anthocyanin. The molar ratio of anthocyanin to acylated casein is 1:7. Dropwise add it while stirring, and dropwise add it at a flow rate of 5 ml / min in a 40 °C water bath and stir magnetically and evenly for 30 min. Then add an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then add the phospholipid powder to ethanol to prepare a 25 mg / mL phospholipid ethanol solution. Ultrasonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution. The mass ratio of phospholipid to acylated casein added is 1:2. Stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then place it under the action of a magnetic field with a magnetic field strength of 1 mT, a time of 7 min, and a temperature of 30 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nano-sustained release delivery system.

[0046] Preparation of Anthocyanin Nanoscale Sustained-Release Delivery System in Example 3

[0047] Dissolve casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to around 7 with PBS, homogenize at 60 °C for 1 h. Add propionic acid as an acylating reagent with a molar ratio of casein to 4:1, and drip at a flow rate of 5 ml / min while stirring magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH to 7. After dialysis and freeze-drying of the reaction solution, take a certain amount of acylated casein and dissolve it in an aqueous solution with ultrasonic assistance. Add 0.01 mol / L HCl solution of anthocyanin, with a molar ratio of anthocyanin to acylated casein of 1:5. Drip and stir simultaneously, and drip at a flow rate of 5 ml / min in a 40 °C water bath while stirring magnetically and evenly for 30 min. Then, add an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then, add phospholipid powder to ethanol to prepare a 20 mg / mL phospholipid ethanol solution, and sonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution, with a molar mass ratio of phospholipid to acylated casein of 1:1, and stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then, place it under the action of a magnetic field with a magnetic field strength of 5 mT, a time of 9 min, and a temperature of 35 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nanoscale sustained-release delivery system.

[0048] Preparation of Anthocyanin Nanoscale Sustained-Release Delivery System in Example 4

[0049] Dissolve the casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to around 7 with PBS, homogenize it at 60 °C for 1 h. Add the acylation reagent propionic acid with a molar ratio of 5:1 to casein, dropwise add it at a flow rate of 5 ml / min and stir magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH = 7. After dialysis of the reaction solution, freeze-dry it. Take a certain amount of acylated casein and ultrasonically assist its dissolution in an aqueous solution. Add anthocyanin diluted with 0.02 mol / L HCl solution, with a molar ratio of anthocyanin to acylated casein of 1:7. Dropwise add it while stirring, and dropwise add it at a flow rate of 5 ml / min in a 40 °C water bath and stir magnetically and evenly for 30 min. Then add an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then add the phospholipid powder to ethanol to prepare a 25 mg / mL phospholipid ethanol solution, sonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution, with a molar mass ratio of phospholipid to acylated casein of 1:2. Stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then place it under the action of a magnetic field, with a magnetic field intensity of 4 mT, a time of 8 min, and a temperature of 40 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nano-sustained release delivery system.

[0050] Preparation of Anthocyanin Nano-Sustained Release Delivery System in Example 5

[0051] Dissolve the casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to around 7 with PBS, homogenize it at 60 °C for 1 h. Add the acylation reagent butyric acid with a molar ratio of 3:1 to casein, dropwise add it at a flow rate of 5 ml / min and stir magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH = 7. After dialysis of the reaction solution, freeze-dry it. Take a certain amount of acylated casein and ultrasonically assist its dissolution in an aqueous solution. Add anthocyanin diluted with 0.01 mol / L HCl solution, with a molar ratio of anthocyanin to acylated casein of 1:4. Dropwise add it while stirring, and dropwise add it at a flow rate of 5 ml / min in a 40 °C water bath and stir magnetically and evenly for 30 min. Then add an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then add the phospholipid powder to ethanol to prepare a 15 mg / mL phospholipid ethanol solution, sonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution, with a molar mass ratio of phospholipid to acylated casein of 1:1. Stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then place it under the action of a magnetic field, with a magnetic field intensity of 2 mT, a time of 10 min, and a temperature of 40 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nano-sustained release delivery system.

[0052] Preparation of Anthocyanin Nanorelease Delivery System in Example 6

[0053] Dissolve casein powder in 0.05 mol / L NaOH solution. After adjusting the pH to about 7 with PBS, homogenize at 60 °C for 1 h. Add butyric acid as the acylation reagent with a molar ratio of butyric acid to casein of 5:1. Dropwise add at a flow rate of 5 ml / min and stir magnetically and evenly for 30 min. Subsequently, add 0.05 mol / L NaOH solution to terminate the reaction and adjust the pH = 7. After dialysis of the reaction solution, freeze-dry it. Take a certain amount of acylated casein and ultrasonically assist in dissolving it in an aqueous solution. Add 0.02 mol / L HCl solution of anthocyanin with a molar ratio of anthocyanin to acylated casein of 1:7. Dropwise add while stirring, and dropwise add at a flow rate of 5 ml / min in a 40 °C water bath and stir magnetically and evenly for 30 min. Then supplement an equal amount of 0.01 mol / L NaOH solution to neutralize the HCl introduced by the anthocyanin solution. Then add phospholipid powder to ethanol to prepare a 25 mg / mL phospholipid ethanol solution. Ultrasonicate for 5 min. After complete dissolution, add it to the acylated casein-anthocyanin complex solution. Add phospholipid with a molar mass ratio of phospholipid to acylated casein of 1:2, and stir magnetically and evenly in a 40 °C water bath for 1 h to fully mix the two. Then place it under the action of a magnetic field with a magnetic field strength of 1 mT, a time of 7 min, and a temperature of 45 °C. Take out the system solution and let it stand at room temperature to obtain the target anthocyanin nanorelease delivery system.

[0054] Morphological Characterization of Anthocyanin Nanorelease Delivery System in Example 7

[0055] After dispersing the anthocyanin nanorelease delivery system prepared in Example 1, perform TEM characterization after negative staining with phosphotungstic acid ( Figure 1 A). It can be seen that the particle size of the prepared nano-system is about 50 - 100 nm. Directly perform TEM characterization on the anthocyanin nanorelease delivery system after preparation ( Figure 1 B). The particle size of the nano-system is also partly between 50 - 100 nm. The observation results of the two are consistent, proving that the particle size range of the nano-system is stable.

[0056] Hydrated Particle Size of Anthocyanin Nanorelease Delivery System in Example 8

[0057] Study dispersing the anthocyanin nanorelease delivery system prepared in Example 1 into a solution before and after freeze-drying, and use a Malvern particle size analyzer to measure the hydrated particle size of the nanoparticles ( Figure 2 ). The particle size of the anthocyanin nano-system is 145 nm, and the PDI is 0.046, indicating that the particle size distribution of the anthocyanin nano-system is relatively uniform and the dispersibility is good.

[0058] Self-Assembly Distribution of Anthocyanin Nanorelease Delivery System in Example 9

[0059] The elemental composition of the anthocyanin nano-sustained release delivery system prepared in Example 1 was analyzed by X-ray photoelectron spectroscopy (XPS). A KRATOS Axis Supra type photoelectron spectrometer was used, with an excitation light source of Al Kα X-ray (energy 1486.6 eV), a power of 400 W, and a base vacuum of 6×10 -9 Torr. The C1s spectral line of extraneous contaminant carbon was used to correct the sample charging effect at 285.0 eV. ESCApe software was used for data processing.

[0060] Table 1. Elemental analysis of CLS nanoparticles

[0061]

[0062] XPS testing is an effective method for detecting the elemental species, chemical states, relative contents, and functional group types on the surface of materials. The XPS elemental analysis results of CLS showed that the content of P element in the CLS surface (0 - 8 nm) was 1.5%. Judging from the experimental raw materials, all of the P element came from phosphatidylcholine, the main component of phospholipids (content 70%), and the P element content of phosphatidylcholine was 1.92%. It can be concluded that the distribution of phosphatidylcholine on the CLS surface was 1.5% / (1.92% × 70%) = 54.7%, that is, more than 50% of the phospholipids in the CLS system were distributed in its 0 - 8 nm surface layer, and the particle size of the CLS system was about 50 nm, further proving that phospholipids would actively distribute on the outer layer of the nano-system and form a "layer-by-layer self-assembly" protection phenomenon of outer layer encapsulation.

[0063] Organ imaging experiment of the anthocyanin nano-sustained release delivery system in Example 10

[0064] Healthy Kunming mice (3 - 4 weeks old, and adaptively fed for 1 week for standby), with a body weight of about 18 - 22 g, were fasted for 12 h before the experiment. Cy5-NHS-labeled CLS was administered by gavage at a dose of 1 mg / kg, and the control group was given the same volume of normal saline; the mice were sacrificed at 0, 2, 4, 8, 12, and 24 h respectively. The heart, liver, spleen, lungs, and kidneys were dissected out, gently rinsed with normal saline to remove the contents, washed with normal saline to remove the residual blood on the surface, blotted dry with filter paper, placed on a glass slide, and directly observed in a live imaging instrument, and fluorescence photos were taken. As Figure 3 shown, after oral administration of Cy5.5-labeled CLS nanoparticles, they were significantly distributed in the lungs and weakly distributed in the liver. The main distribution time in the liver was between 2 - 8 h, and they were distributed in the lungs between 2 - 24 h, especially more in the 2 - 12 h interval. It can be preliminarily speculated that the nano-system has a good lung distribution effect and shows a lung targeting effect.

[0065] Organ tissue distribution experiment of the anthocyanin nano-sustained release delivery system in Example 11

[0066] (1) Take 5 mg of each tissue and organ (heart, liver, spleen, lung, and kidney), and dissolve it in 50 μL of extraction solution (50% methanol aqueous solution containing 0.1% hydrochloric acid).

[0067] (2) Vortex for 5 min, sonicate for 5 min, centrifuge for 3 min (12,000 r / min, 4 °C), and aspirate the supernatant.

[0068] (3) Filter the sample with a microporous membrane (0.22 μm) and store it in a sample vial.

[0069] Chromatographic conditions:

[0070] (1) Instrument: ExionLC TM AD UPLC

[0071] (2) Chromatographic column: ACQUITY BEH C18 1.7 μm, 2.1 mm * 100 mm;

[0072] (3) Mobile phase: Phase A is ultrapure water (0.1% formic acid), and phase B is methanol (0.1% formic acid);

[0073] (4) Elution gradient: The proportion of phase B is 5% at 0.00 min, increases to 50% at 6.00 min, increases to 95% at 12.00 min, is maintained for 2 min, decreases to 5% at 14 min, and is equilibrated for 2 min;

[0074] (5) Flow rate 0.35 mL / min; column temperature 37 °C; injection volume 2 μL.

[0075] Mass spectrometry conditions:

[0076] (1) Instrument: 6500 + MS / MS

[0077] (2) Electrospray ionization source (ESI) temperature 550 °C, mass spectrometry voltage 5500 V in positive ion mode, curtain gas (CUR) 35 psi, declustering potential (DP): 106, and collision energy (CE): 38.

[0078] Further determination of the C3G concentration in the in vivo lung tissue found that at the same time point as the small animal imaging ( Figure 4 ), the enrichment effect of C3G in the lung was consistent with the fluorescence imaging trend, showing an obvious enrichment effect at 2 - 8 h (p < 0.01), while the concentration of C3G monomer was almost zero. This phenomenon suggests that the carrier delivered C3G to the lung and produced an obvious targeted enrichment effect. Comparative analysis of other major organs showed that although the C3G concentration in the CLS system was also higher than that of non-nano C3G, there was no such mutational increase as in the lung tissue. The T of the lung-targeted nano-system 1 / 2= 1.174 h, compared with the T of anthocyanins 1 / 2 = 0.321 h, which is increased by about 3.66 times; the C of the lung-targeted nano-system max = 12.76 nmol / g, compared with the C of anthocyanins max = 2.71 nmol / g, which is increased by about 4.7 times; the AUC of the lung-targeted nano-system (0-t) = 36.3745 nmol / g·h, compared with the AUC of anthocyanins (0-t) = 1.2787 nmol / g·h, which is increased by about 28.4 times.

[0079] Example 12 Improvement experiment of anthocyanin nano-sustained release delivery system on acute lung injury

[0080] As Figure 5 shown, obvious injuries (necrotic tissues, plaques, etc.) appeared in the lungs of mice after acute lung injury modeling, indicating successful modeling ( Figure 5 A). After intervention with anthocyanins, blank carrier (acylated casein + phospholipid) and anthocyanin nano-system, the morphology improved significantly, and the necrotic-like plaques in the tissue decreased, especially in the CLS group. Further analysis of the lung organ index and lung wet / dry ratio found ( Figure 7 ), the organ index and lung wet / dry ratio of the acute lung injury group were significantly higher than those of the blank control group (p < 0.01). After intervention with different groups, only the anthocyanin nano-group showed a downward trend (p < 0.05), indicating that the anthocyanin nano-group has a certain alleviating effect on acute lung injury. HE and Mason morphological analysis found that obvious alveolar wall thickening, a large number of red blood cells, inflammatory cell infiltration, and alveolar fragmentation were observed in the acute lung injury group, which are typical inflammatory case characteristics ( Figure 5 B / C). After treatment with different intervention groups, there was no obvious change in the casein group, a recovery trend appeared in the anthocyanin group, and the inflammatory characteristics of the anthocyanin nano-group were the mildest.

[0081] As Figure 6 shown, compared with the blank group, significant apoptosis occurred in the mice of the acute lung injury group (p < 0.001). After treatment with different intervention groups, the apoptosis of anthocyanins, blank carrier (acylated casein + phospholipid) and anthocyanin nano-system all decreased significantly (p < 0.01), indicating that the three have a protective effect on stem cells. Nrf2 immunohistochemical analysis showed ( Figure 6 B / 7C), compared with the blank group, the expression of Nrf2 in the lung tissue of mice in the acute lung injury group was significantly increased (p < 0.01). After treatment with the intervention group, the expression of Nrf2 was further increased, playing a physiological role in clearing free radicals generated by oxidative stress in the body. Among them, the up-regulation effect of the anthocyanin nano-group was the most significant (p < 0.001). ROS analysis showed ( Figure 6In the acute lung injury group (C / 7E), ROS was significantly increased compared with the blank group (p < 0.01), while after treatment with different intervention groups, a callback was shown (p < 0.01), and there was no significant difference among the three groups. The above research shows that anthocyanins, blank carriers (acylated casein + phospholipids), and anthocyanin nanosystems can effectively resist LPS-induced oxidative stress and apoptosis.

[0082] Figure 8 As shown in A - F, the anthocyanin nanosystem intervention group had a better regulatory effect on inflammatory factors TNF-α, IL-1β, IL-6, IL-10, iNOS, and COX-2 compared with the anthocyanin and blank carrier (acylated casein + phospholipids) groups, indicating that the anthocyanin nanosystem has a better anti-inflammatory effect. Figure 8 As shown in G - J, overall, the anthocyanin nanosystem also had a better regulatory effect on oxidative stress-related enzymes CAT, SOD, GSH, and MDA, indicating that the anthocyanin nanosystem has a better ability to resist oxidative damage in the acute lung injury group compared with anthocyanins and blank carriers (acylated casein + phospholipids).

[0083] Example 13 Improvement Experiment of Anthocyanin Nanodelivery System on Chronic Lung Injury

[0084] As Figure 9 shown, after chronic lung injury modeling, obvious damage (necrosis, plaques, etc.) appeared in the lungs of rats, indicating successful modeling ( Figure 9 A). After intervention with anthocyanins, blank carriers (acylated casein + phospholipids), and anthocyanin nanosystems, the morphology improved significantly, and the necrotic-like plaques in the tissue decreased, especially in the anthocyanin nanosystem group. Further analysis of the lung organ index and lung wet / dry ratio found ( Figure 9 E) that the organ index and lung wet / dry ratio in the chronic lung injury group were significantly increased compared with the blank control group (p < 0.05). After intervention with different groups, only the anthocyanin nanosystem group showed a downward trend (p < 0.05), indicating that the anthocyanin nanosystem group has a certain alleviating effect on chronic lung injury. HE and Mason morphological analyses found that in the chronic lung injury group, typical inflammatory case characteristics such as obvious alveolar wall thickening, a large number of red blood cells and inflammatory cells in the alveoli, and alveolar fragmentation appeared ( Figure 9 B / C). After treatment with different intervention groups, there was no obvious change in the blank carrier group, a recovery trend appeared in the anthocyanin group, and the inflammatory characteristics of the anthocyanin nanosystem were the mildest. Nrf2 immunohistochemical analysis showed ( Figure 9 D / E) that compared with the blank group, the expression of Nrf2 in the lung tissue of rats in the chronic lung injury group was significantly increased (p < 0.001). After treatment with the intervention group, the expression level of Nrf2 was further increased, playing a physiological role in scavenging free radicals generated by oxidative stress in the body, and the up-regulation effect of the anthocyanin nanosystem was the most significant (p < 0.01).

[0085] As Figure 10 shown, compared with the blank group, significant apoptosis was observed in the rats of the chronic lung injury group (p < 0.001). After treatment with different intervention groups, the apoptosis in the anthocyanin, blank carrier (acylated casein + phospholipid), and anthocyanin nanosystem groups decreased significantly (p < 0.01), indicating that all three have a protective effect on stem cells. Further analysis showed that the effect of the anthocyanin nanosystem was significantly better than that of anthocyanin and the blank carrier (p < 0.05). ROS analysis showed ( Figure 10 B / C), the ROS in the chronic lung injury group was significantly higher than that in the blank group (p < 0.001), while after treatment with different intervention groups, anthocyanin (p < 0.01) and the anthocyanin nanosystem (p < 0.001) showed a callback, and the blank carrier group had no obvious effect. The above research indicates that anthocyanin and the anthocyanin nanosystem can effectively resist the oxidative stress and cell apoptosis caused by LPS in chronic lung injury, especially the anthocyanin nanosystem has the best effect.

[0086] Figure 11 A-F show that the anthocyanin nanosystem group has a better regulatory effect on the inflammatory factors TNF-α, IL-1β, IL-6, IL-10, iNOS, and COX-2 compared with the anthocyanin and blank carrier groups, indicating that the anthocyanin nanosystem has a better anti-inflammatory effect. Figure 11 G-J show that overall, the anthocyanin nanosystem also has a better regulatory effect on the oxidative stress-related enzymes CAT, SOD, GSH, and MDA, indicating that the anthocyanin nanosystem has a better effect in resisting oxidative damage caused by chronic lung injury compared with anthocyanin and the blank carrier.

Claims

1. A nanosustained-release delivery system of anthocyanins for oral lung targeting, characterized in that, The preparation method comprises the following steps: Step 1: Preparation of acylated casein. Dissolve casein powder in NaOH solution, adjust the pH to 7, add an acylation reagent, add NaOH solution to terminate the reaction, and after dialysis, freeze-dry to obtain acylated casein for standby. Step 2: Preparation of acylated casein - anthocyanin complex. Take acylated casein and dissolve it in water. After complete dissolution, add dilute HCl solution of anthocyanin, and add NaOH solution to neutralize the HCl introduced by the anthocyanin solution to obtain an acylated casein - anthocyanin complex for standby. The molar ratio of anthocyanin to acylated casein is 1:4 - 1:

7. Step 3: Preparation of anthocyanin nano - sustained release delivery system. Add phospholipid powder into ethanol and ultrasonically dissolve it until completely dissolved. The concentration of the ethanol solution of phospholipid is 15 - 25 mg / mL. Add it to the solution of acylated casein - anthocyanin complex. The mass ratio of phospholipid to acylated casein is 1:1 - 1:

2. Stir, place it under the action of a magnetic field, and self - assemble to form a "molecular cap" - like structure. Take out the system solution and let it stand at room temperature to obtain the anthocyanin nano - sustained release delivery system. The magnetic field strength is controlled between 1 - 5 mT, the magnetization time should be controlled between 5 - 10 min, and the magnetization temperature should be controlled between 25 - 45 °C.

2. The anthocyanin nanosustained release delivery system for oral lung targeting according to claim 1, wherein In Step 1, the acylation reagent is acetic acid or propionic acid or butyric acid, and the molar ratio of the acylation reagent to casein is 3:1 - 5:

1.

3. The anthocyanin nano-sustained release delivery system for oral lung targeting according to claim 1, characterized in that, In Step 1, the concentration of the NaOH solution for dissolving casein is 0.05 mol / L, and the concentration of the NaOH solution for terminating the reaction is 0.05 mol / L. The addition volume stops when the pH = 7.

4. The anthocyanin nano-sustained release delivery system for oral lung targeting according to claim 1, wherein In Step 2, the concentration of the dilute HCl solution is 0.01 - 0.02 mol / L.

5. The anthocyanin nano-sustained release delivery system for oral lung targeting according to claim 1, wherein In Step 3, the stirring is uniform magnetic stirring in a 40 °C water bath for 1 h to fully mix the two.

6. Use of the anthocyanin nano - sustained release delivery system for oral lung targeting described in Claim 1 in the preparation of drugs for treating lung injury.

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