Pericarpium citri reticulatae microcapsule as well as preparation method and application thereof in resisting acute lung injury

Abstract

The invention discloses a pericarpium citri reticulatae microcapsule, a preparation method thereof and application of the pericarpium citri reticulatae microcapsule in resisting acute lung injury. The core material of the pericarpium citri reticulatae microcapsule is the pericarpium citri reticulatae extract, the wall material of the pericarpium citri reticulatae microcapsule is hydroxypropyl-beta-cyclodextrin, and the pericarpium citri reticulatae microcapsule can improve LPS-induced cell injury and peroxidation injury, reduce formation of lipid peroxides, reduce ROS level in cells, reduce MDA content and increase SOD and CAT activity, has good acute lung injury resistance activity, and can be used for preparing a medicine for treating acute lung injury. The effect of preparing the medicine for resisting acute lung injury is better than that of the pericarpium citri reticulatae extract prepared by the traditional extraction method and the micro gum of the pericarpium citri reticulatae extract. Meanwhile, the tangerine peel microcapsule provided by the invention is simple and quick in preparation method, can improve the stability of the tangerine peel extract, has the advantages of sustained release, good water solubility, small irritation, high safety and the like, can relieve lung inflammation and promote lung function recovery, has very good acute lung injury resisting activity, and can be used for preparing the oral preparation. The traditional Chinese medicine composition is used for preparing more natural medicines for resisting acute lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to a tangerine peel microcapsule, a preparation method thereof, and an application thereof in preventing acute lung injury. Background Art

[0002] Acute lung injury (ALI) is a disease caused by various direct and indirect factors that damage alveolar epithelial cells and capillary endothelial cells, resulting in diffuse interstitial and alveolar edema, leading to acute hypoxic respiratory insufficiency, and then triggering a series of inflammatory reactions. The pathophysiological characteristics are reduced lung volume, decreased lung compliance, and ventilation / perfusion imbalance. Clinically, it manifests as progressive hypoxemia and respiratory distress. Lung imaging shows non-uniform exudative lesions, which develop to a severe stage. Clinically, it can cause a variety of diseases, and there are also many diseases caused by ALI, such as pneumonia, severe sepsis, gastroesophageal reflux, fat and amniotic fluid embolism, pancreatitis, pulmonary contusion, reperfusion injury, etc.

[0003] Most of the current clinical drugs for the treatment of acute lung injury are glucocorticoids, which have anti-inflammatory effects and can be used to treat acute lung injury caused by lung infection, such as dexamethasone injection, prednisolone acetate injection, etc. However, the frequent use of glucocorticoids can cause side effects such as inducing or aggravating infections, cardiovascular system complications or digestive system complications. Long-term use of glucocorticoids can cause sodium and water retention, increased blood lipids, and can also lead to hypertension and atherosclerosis; long-term use can aggravate gastric or duodenal ulcers, and side effects are more likely to occur when used in combination with other gastric irritants and drugs. A small number of patients may also develop pancreatitis or fatty liver. Therefore, there is still a lack of natural drugs that can replace glucocorticoids, have no side effects, and have better safety for the treatment of acute lung injury.

[0004] At present, more and more studies have shown that the use of natural Chinese medicine formulas has a good therapeutic effect on acute lung injury. For example, a Chinese medicine composition disclosed in the prior art includes the following raw materials: Paris polyphylla, Scutellaria baicalensis, Fritillaria thunbergii, Commelina communis, Rhizoma Anemarrhenae, Gypsum, Tangerine peel, Citrus aurantium, Fructus Xanthii, Apricot kernel, Platycodon grandiflorum, Pogostemon cablin, Folium Perillae, Radix Glycyrrhizae, which can be used to prepare drugs for the treatment of acute lung injury. However, the pure Chinese medicine composition disclosed in the prior art is to combine multiple Chinese medicine ingredients to work in a synergistic manner. The Chinese medicine ingredients are numerous and complex, the time for boiling the medicine is long, and the components do not play the best effect. The utilization of the active substances of Chinese medicine in the composition also needs to be improved, which affects the efficacy of the medicine and has a high cost.

[0005] The Chinese herbal medicine tangerine peel is a medicine for regulating qi. It is the dried and mature peel of the citrus plant and its cultivated varieties of the Rutaceae family. Tangerine peel is pungent and fragrant, bitter and dry, and warming. It enters the spleen and lung meridians. It regulates the rise and fall of the spleen and lung qi and regulates qi and regulates the middle, and it also dries dampness and regulates qi and resolves phlegm. It can be used for all symptoms of qi stagnation, dampness obstruction, and phlegm congestion. It is especially good for treating qi stagnation in the middle burner, and is most suitable for those with cold. At present, there are few active drugs prepared from tangerine peel alone for the treatment of acute lung injury. Therefore, it is urgent to develop more acute lung injury treatment drugs prepared from the single natural Chinese herbal medicine tangerine peel with better therapeutic effects. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing traditional Chinese medicines for treating pneumonia, and to provide a tangerine peel microcapsule and a preparation method thereof and an application thereof in resisting acute lung injury.

[0007] The invention aims to provide a tangerine peel microcapsule.

[0008] Another object of the present invention is to provide a method for preparing tangerine peel microcapsules.

[0009] Another object of the present invention is to provide application of tangerine peel microcapsules.

[0010] Another object of the present invention is to provide a medicine.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The invention provides a tangerine peel microcapsule. The core material of the microcapsule is tangerine peel extract, and the wall material is hydroxypropyl-β-cyclodextrin; the mass ratio of the wall material to the core material is (2-5):(1-2); the tangerine peel extract is obtained by extracting tangerine peel with a natural low eutectic solvent; the natural low eutectic solvent is prepared from a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor is 4-methoxybenzaldehyde, and the hydrogen bond donor is selected from one of 5-methyl-2-isopropylphenol, lactic acid, malic acid and maltose.

[0013] The present invention extracts the natural Chinese medicinal material tangerine peel, mixes the tangerine peel powder and a specific natural low eutectic solvent under ultrasound-assisted conditions, heats and stirs evenly, and extracts the tangerine peel extract; then, hydroxypropyl-β-cyclodextrin is used as the core material and the wall material, and a spray drying method is adopted to prepare microcapsules, and the obtained tangerine peel microcapsules have good anti-acute lung injury effect. The hydroxypropyl-β-cyclodextrin used as the wall material in the tangerine peel microcapsule is an etherification product of β-cyclodextrin. By introducing hydroxypropyl into β-cyclodextrin, the intramolecular cyclic hydrogen bond is destroyed, the water solubility is improved, and the lipophilic cavity of cyclodextrin is retained, which has the advantages of strong inclusion capacity, low irritation, safety, etc. The prepared tangerine peel microcapsule particles have good dispersibility, high embedding rate and sustained release effect. The prepared tangerine peel microcapsules were used in an acute lung injury model, which could improve LPS-induced cell damage and peroxidative damage, reduce the formation of lipid peroxides, and reduce ROS levels and MDA content in cells, and increase SOD and CAT activities. They could effectively slow down the damage of LPS to alveolar epithelial cells and had anti-acute lung injury activity. The tangerine peel extract and its microcapsules used to prepare anti-acute lung injury drugs had better effects than the tangerine peel extract and its microcapsules prepared by traditional extraction methods.

[0014] The present invention uses a specific natural deep eutectic solvent to extract tangerine peel and then prepares microcapsules, which can further improve the effect of the core material, and the extraction method has a high yield, and has better anti-acute lung injury activity than the traditional natural deep eutectic solvent extraction; the natural deep eutectic solvent used in the present invention is a low-cost, easy to prepare, biodegradable, sustainable and environmentally friendly at room temperature, non-toxic, and waste-free liquid, which has a strong penetration and erosion ability on the cell wall, and can further improve the extraction efficiency of tangerine peel. In addition, the extraction method provided by the present invention is simple to operate, low in production cost, without any pollution, and high in extraction efficiency, and has a better effect for the preparation of tangerine peel microcapsules.

[0015] Preferably, the mass ratio of tangerine peel to natural deep eutectic solvent is (1-5):(10-20).

[0016] More preferably, the mass ratio of tangerine peel to natural deep eutectic solvent is (1-2): (15-20).

[0017] Preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is (1-5):1.

[0018] More preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1.

[0019] Preferably, the hydrogen bond acceptor is 4-methoxybenzaldehyde, and the hydrogen bond donor is 5-methyl-2-isopropylphenol.

[0020] Further preferably, the mass ratio of the wall material to the core material is 5:1.

[0021] At the same time, the present invention provides a method for preparing tangerine peel microcapsules, comprising the following steps:

[0022] (1) heating and dissolving a hydrogen bond acceptor and a hydrogen bond donor at 60 to 80° C. to prepare a natural deep eutectic solvent;

[0023] (2) Extraction of dried tangerine peel: after mixing dried tangerine peel and a natural deep eutectic solvent, ultrasonic extraction is performed to obtain a dried tangerine peel extract;

[0024] (3) preparing a wall material solution, then adding the core material tangerine peel extract, mixing and stirring to obtain an emulsion; and then spray drying the emulsion to obtain tangerine peel microcapsules.

[0025] Furthermore, the ultrasonic conditions are: extraction temperature 30-70°C, time 20-40min, power 300-440W.

[0026] Preferably, the ultrasonic conditions are: extraction temperature 50°C, time 30min, power 350W.

[0027] Furthermore, the hydrogen bond acceptor and the hydrogen bond donor are heated and dissolved and stirred at a speed of 350 to 500 r / min for 10 to 30 min.

[0028] More preferably, the stirring conditions are: rotation speed 450 r / min, time 30 min.

[0029] Furthermore, the wall material is hydroxypropyl-β-cyclodextrin, and the core material is tangerine peel extract.

[0030] Furthermore, the stirring conditions for mixing the wall material and the core material are: a rotation speed of 10000 to 15000 rpm / min and a time of 10 to 15 min.

[0031] More preferably, the stirring conditions are a rotation speed of 12000 rpm / min and a stirring time of 10 min.

[0032] Furthermore, the inlet temperature of the spray drying method is 120-150° C., the outlet temperature is 45-70° C., and the flow rate is 5-8 mL / min.

[0033] Preferably, the inlet temperature of the spray drying method is 130°C, the outlet temperature is 62°C, and the flow rate is 8 mL / min.

[0034] Furthermore, the sample inlet temperature of the spray drying method is maintained at 20-40°C, and the outlet air temperature is maintained at 70-90°C.

[0035] Preferably, the sample inlet temperature of the spray drying method is maintained at 25°C, and the outlet air temperature is maintained at 80°C.

[0036] The invention provides application of tangerine peel microcapsules in preparing medicines for resisting acute lung injury.

[0037] The invention provides a medicine containing the tangerine peel microcapsule.

[0038] The present invention has the following beneficial effects:

[0039] The tangerine peel microcapsules provided by the invention have good anti-acute lung injury effect, and are used in an acute lung injury model to improve LPS-induced cell damage and peroxidative damage, reduce the formation of lipid peroxides, and reduce the ROS level and MDA content in cells, and increase the activities of SOD and CAT, and can effectively slow down the damage of LPS to alveolar epithelial cells, have anti-acute lung injury activity, and are used for preparing anti-acute lung injury drugs, and have better effects than tangerine peel extracts and microcapsules prepared by traditional extraction methods.

[0040] The preparation method provided by the present invention is simple and fast. After extraction with a natural deep eutectic solvent, there is no need to separate and purify, and microcapsules are directly obtained by spray drying. Through microcapsule drug delivery, hydroxypropyl-β-cyclodextrin is used to form a subject-object interaction through non-covalent bonds, and the tangerine peel extract (object) is wrapped in the hydrophobic cavity of the macrocyclic sugar (subject). The tangerine peel extract can form a stable inclusion compound with hydroxypropyl-β-cyclodextrin, improve the characteristics of the stability of the tangerine peel extract, and can also be continuously released, with a good sustained release effect. Hydroxypropyl-β-cyclodextrin is selected as the wall material, which improves the water solubility and stability of the microcapsule, and has the advantages of strong inclusion capacity, low irritation, and safety. The microcapsule prepared by the present invention can greatly improve the stability of the drug, and has a constant release drug characteristic, can alleviate lung inflammation, promote lung function recovery, and has a good anti-acute lung injury activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 These are scanning electron micrographs of tangerine peel microcapsules (a is hydroxypropyl-β-cyclodextrin microcapsules; b is sodium alginate microcapsules; c is chitosan microcapsules).

[0042] Figure 2 This is the particle size distribution diagram of microcapsules.

[0043] Figure 3 This is the release curve of tangerine peel extract microcapsules.

[0044] Figure 4 This is the effect of tangerine peel microcapsules on the survival rate of A549 cells.

[0045] Figure 5 The effect of different concentrations of LPS on the survival rate of A549 cells.

[0046] Figure 6 In vitro anti-acute lung injury activity of tangerine peel microcapsules.

[0047] Figure 7 This is the effect of tangerine peel microcapsules on MDA content, SOD and CAT enzyme activities in acute lung injury cell model.

[0048] Figure 8 The effect of tangerine peel microcapsules on the reactive oxygen content in an acute lung injury cell model. DETAILED DESCRIPTION

[0049] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0050] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0051] Example 1 Selection of raw materials

[0052] 1. Selection of natural deep eutectic solvents

[0053] According to the ratio set in Table 1, different hydrogen bond donors 5-methyl-2-isopropylphenol and hydrogen bond acceptor 4-methoxybenzaldehyde are respectively mixed, and heated and stirred at 80°C until the prepared liquid solvent is uniform to obtain a low eutectic solvent. Dry, non-mildewed high-quality tangerine peel is selected again, and after washing the foreign matter in the tangerine peel with clear water, it is dried and crushed into powder. Take tangerine peel powder and different low eutectic solvents, extract at a mass ratio of 1:20, at an ultrasonic power of 350W, a time of 30min, and an extraction temperature of 50°C. The extraction rate of different extracts is calculated according to the following formula, and the content of active substance flavonoids in the tangerine peel extract prepared above is measured by ultra-high performance liquid chromatography (LCMS).

[0054] Yield = (extract mass / tangerine peel powder mass) × 100%

[0055] Table 1 Formulas of different natural deep eutectic solvents

[0056]

[0057]

[0058] The measurement results are shown in Table 1, which show that under the same other conditions, the extraction efficiency of the tangerine peel extract obtained by extracting the hydrogen bond acceptor and hydrogen bond donor using 4-methoxybenzaldehyde and 5-methyl-2-isopropylphenol is the highest, and the content of active flavonoids is also relatively high, reaching 14.46 mg / g.

[0059] 2. Selection of microcapsule wall materials and structural performance analysis

[0060] The wall materials of the microcapsules are hydroxypropyl-β-cyclodextrin, sodium alginate and chitosan, respectively, and the microcapsules are prepared by spray drying. The above wall materials are added to deionized water, heated and stirred to dissolve, and a wall material solution is formed. The core material tangerine peel extract (using DESs-1 group, the mass ratio of wall material to core material is 5:1) and food coloring (for color determination) are added and mixed and stirred until uniform, and the stirring speed is 10000-15000rpm / min, 10-15min, and an emulsion is obtained. The emulsion is then spray dried into powder. The inlet temperature of the spray drying method is 130℃, the outlet temperature is 62℃, the sample inlet temperature is maintained at 25℃, the outlet air temperature is maintained at 80℃, and the flow rate is 8mL / min. The tangerine peel extract microcapsules are obtained.

[0061] (1) Structural analysis: Microcapsules made of hydroxypropyl-β-cyclodextrin, sodium alginate, and chitosan were tested by scanning electron microscopy. The samples were fixed on the sample stage with conductive double-sided tape, and the excess sample was blown away. After gold spraying, the samples were observed under 15 kV and the magnification was 700 times.

[0062] The results are as follows Figure 1 As shown, Figure 1 a represents the SEM image of hydroxypropyl-β-cyclodextrin microcapsules. It can be seen from the figure that the microcapsule particles have good dispersion and no adhesion occurs. Figure 1 b shows the SEM image of sodium alginate microcapsules. It can be seen from the figure that the microcapsules have some holes, which is not conducive to embedding. Figure 1 c represents the SEM image of chitosan microcapsules. It can be seen from the figure that the microcapsules have depressions, which are more unfavorable for embedding.

[0063] (2) Determination of embedding rate: Weigh a certain amount of tangerine peel extract and dissolve it in the microcapsule aqueous solution. Determine the polyphenol content of the tangerine peel extract in the solution before embedding and the polyphenol content of the tangerine peel extract in the centrifugal supernatant after embedding. Determine the polyphenol content in the solution according to the gallic acid method. Calculate the microcapsule embedding rate according to the following formula:

[0064] Q=A / B×100%

[0065] Wherein: Q is the microcapsule embedding rate, %; A is the polyphenol content of tangerine peel extract in the supernatant, g / mL; B is the polyphenol content of tangerine peel extract in the solution before embedding, g / mL.

[0066] The measurement results are shown in Table 2, which show that when the mass ratio of the wall material to the core material is 5:1, the wall material using hydroxypropyl-β-cyclodextrin has a higher embedding rate for the tangerine peel extract.

[0067] Table 2 Embedding efficiency of microcapsules prepared with different wall materials

[0068] Wall material Embedding rate (%) Hydroxypropyl-β-cyclodextrin 89.59 Sodium alginate 88.88 Chitosan 86.24

[0069] (3) Water solubility: The prepared microcapsules were then placed in distilled water and the color changes in the water were observed. The results showed that the color of the wall material using hydroxypropyl-β-cyclodextrin was darker, indicating good water solubility. The color of the aqueous solution of the wall material of sodium alginate and chitosan was lighter, indicating poor water solubility. Therefore, hydroxypropyl-β-cyclodextrin was selected as the wall material. Hydroxypropyl-β-cyclodextrin is an etherification product of β-cyclodextrin. By introducing hydroxypropyl into β-cyclodextrin, the cyclic hydrogen bonds in the molecule are destroyed, thereby improving the water solubility. At the same time, the lipophilic cavity of cyclodextrin is retained, which has the advantages of strong inclusion capacity, low irritation, and safety.

[0070] (4) Particle size determination: Weigh 1 g of hydroxypropyl-β-cyclodextrin microcapsules into a beaker, add pure water, and perform ultrasonic treatment for 10 min to completely break them. Take the supernatant into a glass dish and measure the particle size distribution using a particle size distribution analyzer.

[0071] The results are as follows Figure 2 As shown, the particle size of the hydroxypropyl-β-cyclodextrin microcapsules is uniform, and the particle size distribution is about 10 μm.

[0072] 3. Sustained release performance test of microcapsules

[0073] (1) Tangerine peel extract / PSB standard curve drawing: Using phosphate buffered saline (PBS) with a pH of 7.4 as a sustained-release system for tangerine peel extract, tangerine peel extract / PBS standard solutions with concentrations of 1.2, 1.0, 0.8, 0.6, 0.4, and 0.2 mg / mL were prepared, and the maximum absorption peak at 400 nm was detected by ultraviolet spectrophotometer to draw a tangerine peel extract / PBS standard curve under the sustained-release system. The regression equation of tangerine peel extract in PBS solution was obtained experimentally as follows:

[0074] Y=0.44648X+0.02342(R 2 =0.9999)

[0075] Wherein, X is the content of tangerine peel extract (mg), and Y is the ultraviolet absorbance corresponding to the flavonoid peak spectrum.

[0076] (2) Test of sustained-release performance of tangerine peel extract microcapsules / PBS: Use a measuring cylinder to measure 20 mL of PBS buffer and put it into a beaker. Then accurately weigh 10 mg of tangerine peel extract microcapsules (wall materials are hydroxypropyl-β-cyclodextrin, sodium alginate, and chitosan, respectively), add them to the beaker, and place it in a 37°C water bath thermostat. Stir magnetically for 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 16 h, then draw 4 mL of supernatant, add the same volume of buffer, measure the absorbance at 400 nm with a UV spectrophotometer, substitute it into the tangerine peel extract / PBS standard curve equation, calculate the solution concentration, then calculate the mass of tangerine peel extract released, and finally calculate the sustained-release rate of the tangerine peel extract microcapsules.

[0077] The calculation formula of sustained release rate: Q T =(C T *V0+∑C T-1 *V) / m

[0078] m: actual mass of drug loaded in gel microspheres

[0079] Q T : Cumulative release rate at sampling time T

[0080] C T : Drug concentration in the solution when sampling at time T

[0081] C T-1 : Drug concentration in the solution when sampling at time T-1

[0082] V0: Volume of the release system

[0083] V: volume at the time of sampling

[0084] The results are as follows Figure 3 As shown in the figure, it can be seen that as the sustained-release time is prolonged, the cumulative release amount of the dried tangerine peel extract gradually increases. The dried tangerine peel extract concentration in the PBS solution of the hydroxypropyl-β-cyclodextrin microcapsule no longer changes at about 12 hours, indicating that the dried tangerine peel extract has been completely released, and the maximum sustained-release amount of the dried tangerine peel extract is 79.01%, with a good sustained-release effect; while the dried tangerine peel extract concentration in the PBS solution of the sodium alginate microcapsule and the chitosan microcapsule no longer changes at about 10 hours, and their maximum sustained-release amounts of the dried tangerine peel extract are 62.21% and 64.52%, respectively, and their sustained-release effects are poor.

[0085] Example 2 Preparation of Tangerine Peel Microcapsules

[0086] (1) Preparation of natural deep eutectic solvents (NADESs): 4-methoxybenzaldehyde and 5-methyl-2-isopropylphenol were mixed in a molar ratio of 1:1 and heated, and stirred continuously at 80°C until the liquid solvent was uniform.

[0087] (2) Extraction of dried tangerine peel: Select high-quality dried tangerine peel without mildew, wash away foreign matter in the tangerine peel with clean water, dry and crush into powder. Take tangerine peel powder and natural deep eutectic solvent at a mass ratio of 1:15, perform ultrasonic assisted extraction, extract at 50°C, ultrasonic power 350W, time 30min, and obtain tangerine peel extract.

[0088] (3) Preparation of microcapsules: Add hydroxypropyl-β-cyclodextrin as a wall material into deionized water, heat and stir to dissolve, and form a wall material solution; add tangerine peel extract as a core material into the wall material solution at a mass ratio of 5:1, mix and stir until uniform, and obtain an emulsion; then spray dry the emulsion to obtain a powder, namely the tangerine peel extract microcapsules.

[0089] Example 3 Preparation of Tangerine Peel Microcapsules

[0090] (1) Preparation of natural deep eutectic solvents (NADESs): 4-methoxybenzaldehyde and 5-methyl-2-isopropylphenol were mixed in a molar ratio of 1:1 and heated, and stirred continuously at 80°C until the liquid solvent was uniform.

[0091] (2) Extraction of dried tangerine peel: Select high-quality dried tangerine peel without mildew, wash away foreign matter in the tangerine peel with clean water, dry and crush into powder. Take tangerine peel powder and natural deep eutectic solvent at a mass ratio of 1:10, perform ultrasonic assisted extraction, extract at 50°C, ultrasonic power 350W, time 30min, and obtain tangerine peel extract.

[0092] (3) Preparation of microcapsules: Add hydroxypropyl-β-cyclodextrin as a wall material into deionized water, heat and stir to dissolve, and form a wall material solution; add tangerine peel extract as a core material into the wall material solution at a mass ratio of 5:1, mix and stir until uniform, and obtain an emulsion; then spray dry the emulsion to obtain a powder, namely the tangerine peel extract microcapsules.

[0093] Example 4

[0094] The preparation method of tangerine peel microcapsules in this embodiment is the same as that in embodiment 2, except that the ultrasonic power for tangerine peel extraction is 300W.

[0095] Example 5

[0096] The preparation method of tangerine peel microcapsules in this embodiment is the same as that in embodiment 2, except that the ultrasonic power for tangerine peel extraction is 400W.

[0097] Example 6

[0098] The preparation method of tangerine peel microcapsules in this embodiment is the same as that in embodiment 2, except that the ultrasonic time for tangerine peel extraction is 20 min.

[0099] Example 7

[0100] The preparation method of tangerine peel microcapsules in this embodiment is the same as that in embodiment 2, except that the ultrasonic time for tangerine peel extraction is 40 min.

[0101] Comparative Example 1 Traditional natural deep eutectic solvent extraction method

[0102] The tangerine peel extraction method of this comparative example is the same as that of Example 1, except that the natural deep eutectic solvent is prepared from choline chloride-urea (molar ratio 1:1), and the filtrate is filtered after ultrasonic extraction, and the filtrate is concentrated under reduced pressure to 70 mL to obtain.

[0103] Comparative Example 2 Traditional natural deep eutectic solvent extraction method

[0104] The tangerine peel extraction method of this comparative example is the same as that of Example 1, except that the natural low eutectic solvent is prepared from choline chloride-lactic acid (molar ratio 1:1), and the filtrate is filtered after ultrasonic extraction, and the filtrate is concentrated under reduced pressure to 70 mL to obtain.

[0105] Comparative Example 3 Preparation of Traditional Tangerine Peel Microcapsules

[0106] The preparation method of the tangerine peel microcapsules in this comparative example is the same as that in Example 2, except that the tangerine peel extract as the core material is the extract prepared in Comparative Example 1.

[0107] Comparative Example 4: No microcapsule loading

[0108] The tangerine peel extract prepared by the DESs-1 group in the example was not microencapsulated.

[0109] Test Example 1 Effect of different extraction conditions on the extraction of tangerine peel extract

[0110] The yields of the tangerine peel extracts obtained before the microcapsule preparation in Examples 2 to 7 were measured by the same method as in Example 1. The results are shown in Table 3, showing that the yield of the tangerine peel extract obtained by the extraction conditions of Example 1 is the best. In Examples 4 to 7, it is shown that the ultrasonic conditions will affect the extraction rate of the tangerine peel extract, but the extraction effect is still better than that of Comparative Examples 1 to 2. The lifting efficiency of the tangerine peel extract is greatly affected by adjusting the mass ratio of tangerine peel and natural deep eutectic solvent.

[0111] Table 3 Extraction effects of different extraction conditions

[0112]

[0113] Test Example 2 Cytotoxicity Detection

[0114] 1. Human alveolar epithelial cell culture

[0115] A549 cells (human alveolar epithelial cells) retain the important characteristics of type II alveolar epithelial cells and are a commonly used cell line for studying acute lung injury. A549 cells were cultured in a complete culture medium (containing 90% DMEM culture medium, 10% fetal bovine serum and 1% penicillin-streptomycin double antibody solution) at 37°C, saturated humidity, and a culture incubator containing 5% CO2; when the cells grew to basically cover the bottom of the culture bottle, the waste liquid in the cell culture bottle was removed, and after washing with PBS, 1mL 0.25% trypsin was added for digestion for 1min, and 2mL complete culture medium was added to terminate the reaction, and the bottom of the bottle was blown with a pipette and transferred to a new centrifuge tube, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and an appropriate amount of complete culture medium was added to resuspend, and subculture was performed at a ratio of 1:2. The experiment was carried out after the cell growth state stabilized.

[0116] 2. Cytotoxicity assay

[0117] A549 cells were cultured at 5×10 4 The cells were inoculated in a 96-well plate at 100 μL per well and divided into a blank group (culture solution containing cells), an experimental group (culture solution containing cells and tangerine peel extract microcapsules of different concentrations prepared by the DESs-1 group in Example 1 (the wall material is hydroxypropyl-β-cyclodextrin)) and a cell-free zeroing group, and 3 replicate wells were set for each group; after incubation for 24 h, the supernatant was discarded, and the experimental group was treated with 100 μL of DMEM culture medium containing tangerine peel extract microcapsules of different concentrations (10, 50, 100 and 500 μg / mL), and the other groups were treated with 100 μL of DMEM culture medium. After culturing for 24 h, 10 μL of CCK8 was added to each well, and the absorbance was measured at 450 nm after incubation for 1.5 h, and the cell survival rate (%) was calculated according to the following formula.

[0118] Cell survival rate = (OD experimental group - OD zero adjustment group) / (OD blank group - OD zero adjustment group) × 100%

[0119] Effects of different concentrations of tangerine peel microcapsules on the survival rate of A549 cells Figure 4 As shown in the figure, it can be seen that in the concentration range of 10-100 μg / mL, the tangerine peel extract exhibits a non-toxic effect. Therefore, the present invention uses 100 μg / mL as the intervention concentration of the tangerine peel microcapsule for subsequent experiments.

[0120] Test Example 3 Anti-acute lung injury activity detection

[0121] 1. Establishment of acute lung injury cell model

[0122] A549 cells were cultured at 5 × 10 4 / mL, 100 μL per well was inoculated in a 96-well plate and divided into a blank group and a lipopolysaccharide group (LPS group), and 3 replicates in each group were cultured for 24 hours; the supernatant was discarded, and 100 μL of DMEM medium was added to make the LPS group contain 1, 5, 10, 15, and 20 μg / mL LPS, respectively; the culture was continued for 24 hours, and the survival rate of each group of cells was detected according to the method described in Test Example 2. The cell survival rate reflected the degree of LPS-induced cell damage, so as to screen the appropriate modeling concentration.

[0123] Effects of different concentrations of LPS on the survival rate of A549 cells Figure 5 As shown in the figure, when the LPS concentration was 10-20 μg / mL, the cell survival rate decreased significantly compared with the normal group (p<0.05). The acute lung injury cell model was established with a LPS concentration of 20 μg / mL corresponding to a survival rate of about 60%.

[0124] 2. Intervention effects of each group on the acute lung injury cell model

[0125] A549 cells were cultured at 5 × 10 4 / mL, 100 μL per well was inoculated in a 96-well plate and divided into a normal group, an LPS group, an tangerine peel microcapsule group in Example 1, a comparative example 3 group, and a comparative example 4 group. After incubation for 24 hours, the supernatant was discarded, 100 μL DMEM medium was added to the normal group, and 100 μL of 100 μg / mL tangerine peel microcapsule solution was added to different tangerine peel microcapsule groups, and the final concentration of LPS in the LPS group and the tangerine peel microcapsule group was 20 μg / mL; after continuing to culture for 24 hours, the survival rate of each group of cells was detected according to the method described in Test Example 2, and the cell survival rate reflected the in vitro anti-acute lung injury activity of the tangerine peel extract microcapsule.

[0126] In vitro anti-acute lung injury activity of different tangerine peel microcapsules Figure 6 As shown in the figure, it can be seen that the tangerine peel microcapsules of Example 1 have a significant improvement on the survival rate of the acute lung injury cell model cells (p<0.05 or p<0.01). The effect is better than that of Comparative Examples 3 and 4, that is, the tangerine peel microcapsules prepared by the present invention have an improvement effect on LPS-induced cell damage and have anti-acute lung injury activity.

[0127] 3. Effects on MDA content, SOD and CAT enzyme activities in acute lung injury cell model

[0128] A549 cells were cultured at 5 × 10 5The number of cells / well was inoculated into 6-well culture plates, and the supernatant was discarded after culturing for 24 h, and the plates were washed with PBS; 1 mL of DMEM culture medium was added to the normal group, 1 mL of 100 μg / mL concentration tangerine peel microcapsule solution was added to the tangerine peel microcapsule group of Example 1, 1 mL of 100 μg / mL concentration tangerine peel microcapsule solution was added to the comparative example 3 group, and 1 mL of 100 μg / mL concentration tangerine peel microcapsule solution was added to the comparative example 4 group, and the final concentration of LPS in the LPS group and the embodiment 1 group, the comparative example 3 group, and the comparative example 4 group was 20 μg / mL, and cells were collected after continuing to culture for 24 h, and the MDA content, SOD and CAT activity in each group of cells were measured according to the instructions of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) detection kits (purchased from Suzhou Ge Rui Si Biotechnology Co., Ltd.), and the protein concentration was measured with a BCA kit (purchased from Biyuntian Biotechnology Research Institute).

[0129] Effects of each group on MDA content, SOD and CAT enzyme activities in acute lung injury cell model Figure 7 As shown in the figure, compared with the blank group, the MDA content in the model group cells increased significantly, and the SOD and CAT enzyme activities decreased (P<0.01); compared with the model group, the tangerine peel microcapsule group in Example 1 can significantly reduce the MDA content (P<0.05 or P<0.01), and can significantly increase the SOD and CAT activities. Compared with the group in Example 1, the MDA of Comparative Examples 3 and 4 is significantly increased, and the SOD and CAT activities are significantly reduced. It shows that the tangerine peel microcapsules in Example 1 can improve the peroxidative damage in the acute lung injury cell model to a certain extent and reduce the formation of lipid peroxides.

[0130] 4. Effects on reactive oxygen species content in acute lung injury cell model

[0131] A549 cells were cultured at 1×10 4 The cells were inoculated in 96-well plates in the number of cells / well and divided into a normal group, an LPS group, and an tangerine peel microcapsule group of Example 1, a comparative example 3 group, and a comparative example 4 group. After 24 hours of cultivation, the supernatant was discarded, 100 μL of DMEM culture medium was added to the normal group, 100 μL of 100 μg / mL concentration tangerine peel microcapsule solution was added to the tangerine peel microcapsule group of Example 1, 100 μL of 100 μg / mL concentration tangerine peel microcapsule solution was added to the comparative example 3 group, and 100 μL of 100 μg / mL concentration tangerine peel extract solution was added to the comparative example 4 group, and the final concentration of LPS in the LPS group and the embodiment 1 group, comparative example 3, and comparative example 4 was 20 μg / mL, and the cultivation was continued for 24 hours; the cellular active oxygen (ROS) level was measured according to the instructions of the active oxygen detection kit (purchased from Biyuntian Biotechnology Research Institute), and the intracellular ROS content was indicated by the size of the fluorescence intensity value.

[0132] ROS is a substance that can be produced by normal or abnormal cell metabolism. Excessive ROS can destroy the normal function of biofilms and cells, reduce enzyme activity, and promote cell death. Figure 8 As shown. As can be seen from the figure, compared with the blank group, the ROS level in the model group cells was significantly increased (P<0.01), and under the action of the tangerine peel microcapsules in Example 1, the ROS level was significantly reduced (P<0.01). Combined with the results of the MDA content, SOD and CAT enzyme activity in the cells, it can be seen that under the induction of LPS, the ROS level in the cells increased, the content of the ROS metabolite MDA increased, and the activity of SOD and CAT that inhibited the production of ROS decreased. Under the action of the tangerine peel microcapsules in Example 1, the ROS level in the cells decreased, the MDA content decreased, and the SOD and CAT activities increased, which can effectively slow down the damage of LPS to alveolar epithelial cells, and has better effects than Comparative Examples 3 to 4.

[0133] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A tangerine peel microcapsule, characterized in that: The core material of the microcapsule is tangerine peel extract, and the wall material is hydroxypropyl-β-cyclodextrin; the mass ratio of the wall material to the core material is (2-5): (1-2); The tangerine peel extract is obtained by extracting tangerine peel with a natural low eutectic solvent; the natural low eutectic solvent is prepared from a hydrogen bond acceptor and a hydrogen bond donor; the hydrogen bond acceptor is 4-methoxybenzaldehyde, and the hydrogen bond donor is selected from one of 5-methyl-2-isopropylphenol, lactic acid, malic acid, and maltose.

2. The tangerine peel microcapsule according to claim 1, characterized in that: The mass ratio of the tangerine peel to the natural deep eutectic solvent is (1-5): (10-20).

3. The tangerine peel microcapsule according to claim 2, characterized in that: The mass ratio of the tangerine peel to the natural deep eutectic solvent is (1-2): (15-20).

4. The tangerine peel microcapsule according to claim 2, characterized in that: The molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is (1-5):

1.

5. The tangerine peel microcapsule according to claim 4, characterized in that: The hydrogen bond acceptor is 4-methoxybenzaldehyde, and the hydrogen bond donor is 5-methyl-2-isopropylphenol.

6. The method for preparing the tangerine peel microcapsules according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) heating and dissolving a hydrogen bond acceptor and a hydrogen bond donor at 60 to 80° C. to prepare a natural deep eutectic solvent; (2) Extraction of dried tangerine peel: after mixing dried tangerine peel and a natural deep eutectic solvent, ultrasonic extraction is performed to obtain a dried tangerine peel extract; (3) preparing a wall material solution, then adding the core material tangerine peel extract, mixing and stirring to obtain an emulsion; and then spray drying the emulsion to obtain tangerine peel microcapsules.

7. The method according to claim 6, characterized in that: The ultrasonic extraction conditions in (2) are as follows: temperature 30-70°C, power 300-440W, and time 20-40min.

8. The method according to claim 6, characterized in that: The inlet temperature of spray drying is 120-150°C, and the outlet temperature is 45-70°C; the sample inlet temperature is maintained at 20-40°C, the outlet air temperature is maintained at 70-90°C, and the flow rate is 5-8 mL / min.

9. Use of the tangerine peel microcapsule according to claim 1 in the preparation of a drug for resisting acute lung injury.

10. A drug, characterized in that Containing the tangerine peel microcapsule according to claim 1.

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