Preparation method of esterified product of oil-tea camellia shell, ester group composite microspheres of oil-tea camellia shell, and preparation method and application thereof

Abstract

The present invention discloses a preparation method of an esterification product of camellia oleifera fruit shell: The camellia oleifera fruit shell is crushed and sieved, extracted in a mixed solvent of toluene and ethanol, and the residue is vacuum dried to a constant weight. An ionic liquid is added, and the camellia oleifera shell is completely dissolved by stirring under nitrogen protection to obtain a camellia oleifera shell solution; then acetic anhydride is added, and the reaction is carried out under nitrogen protection. The reaction mixture is poured into ethanol to terminate the reaction, centrifuged and separated, and the solid after the reaction is collected and vacuum dried to a constant weight to obtain a camellia oleifera fruit shell acetic anhydride esterification product. The present invention also discloses a camellia oleifera fruit shell ester-based composite microsphere based on the above camellia oleifera fruit shell esterification product, its preparation method and application. The preparation method of the camellia oleifera fruit shell esterification product of the present invention improves the solubility of the camellia oleifera fruit shell and has excellent processing performance. The camellia oleifera fruit shell ester-based composite microsphere has a colon-targeting effect, can improve the stability and bioavailability of tea polyphenols, has good antibacterial and anti-inflammatory effects, can regulate intestinal immune responses, and treat intestinal inflammation.

Description

Technical Field

[0001] The present invention relates to the field of medical drug carrier materials, and particularly relates to a preparation method of an esterification product of camellia oleifera shell, an ester-based composite microsphere of camellia oleifera shell and a preparation method and application thereof. Background Art

[0002] It is reported that camellia oleifera shell contains various active ingredients such as flavonoids, polyphenols, polysaccharides, tea saponins, tannins, etc., and has pharmacological effects such as antibacterial, anti-inflammatory, antioxidant, and immunomodulatory effects. The camellia oleifera shell fiber mainly contains cellulose, hemicellulose and lignin, which is a natural polymer material with a complex structure, and has the advantages of wide source, renewable, low cost, good biocompatibility, biodegradability, etc. Making full use of this part of lignocellulosic biomass is of great significance for improving the added value of camellia oleifera.

[0003] Cellulose is an environmentally friendly material with rich sources, easy to modify and good biocompatibility, and has been prepared into various forms of drug carriers such as microspheres, hydrogels, and aerogels. However, most current studies mainly focus on extracting cellulose from biomass, resulting in a large amount of hemicellulose and lignin being discarded (Chen Z, Xu H N, Ouyang X K. The Simultaneous Production of Two Distinct Types of Cellulose Nanocrystals, Langmuir: The ACS Journal of Surfaces and Colloids, 2022(19):38). In addition, the separation of cellulose usually involves a large amount of toxic chemicals and environmental pollution, with low efficiency and is not suitable for large-scale industrial production (Avinash P. Manian, Michael Cordin, Tung Pham. Extraction of cellulose fibers from flax and hemp: a review. Cellulose, 2021, 28(13):8275-8294). If a complete dissolution system of lignocellulose is used to directly develop lignocellulosic biomass into a carrier material, the complex separation process and pollution problems of lignocellulose components can be avoided, the production cost can be reduced, and it is beneficial to realize the comprehensive high-value utilization of lignocellulosic biomass.

[0004] Tea polyphenols are the general term for polyhydroxyphenolic substances in tea, and play an important role in antibacterial, anti-inflammatory, antioxidant, anti-apoptosis and inhibiting the growth of cancer cells, and are widely used in the fields of food, daily chemicals and medicine. However, the poor oral stability and low bioavailability of tea polyphenols greatly limit the application of tea polyphenols. Summary of the Invention

[0005] To overcome the above-mentioned drawbacks and deficiencies of the prior art, one of the objectives of the present invention is to provide a method for preparing an esterification product of camellia oleifera fruit shell, which performs homogeneous acetic anhydride esterification on the camellia oleifera fruit shell, not only effectively improving the solubility of the camellia oleifera fruit shell and endowing it with excellent processing performance, but also being beneficial to the loading of drugs.

[0006] Another objective of the present invention is to provide a camellia oleifera fruit shell ester-based composite microsphere, which has the characteristics of uniform particle size, high encapsulation rate and drug loading rate, good antibacterial and anti-inflammatory effects, good biocompatibility, and biodegradability.

[0007] A third objective of the present invention is to provide a method for preparing a camellia oleifera fruit shell ester-based composite microsphere, which is easy to realize industrial production.

[0008] A fourth objective of the present invention is to provide the application of the above-mentioned camellia oleifera fruit shell ester-based composite microsphere, which can simultaneously exert antibacterial and anti-inflammatory functions, regulate intestinal immune responses and modulate the gut microbiome, thereby treating intestinal inflammation, and at the same time has a certain colon-targeting effect, can deliver drugs to the inflammatory site, and improve the treatment effect.

[0009] The objectives of the present invention are achieved through the following technical solutions:

[0010] A method for preparing an esterification product of camellia oleifera fruit shell, comprising the following steps:

[0011] Crush and sieve the camellia oleifera fruit shell, extract it in a mixed solvent of toluene and ethanol at 70 - 90 °C for 4 - 6 h, dry the residue under vacuum to constant weight, add an ionic liquid, stir at 100 - 110 °C for 4 - 6 h under nitrogen protection to completely dissolve the camellia oleifera shell, and obtain a camellia oleifera shell solution; then add acetic anhydride, react at 90 - 100 °C for 90 - 120 min under nitrogen protection, pour the reaction mixture into ethanol to terminate the reaction, perform centrifugal separation, collect the solid after the reaction, and dry it under vacuum to constant weight to obtain an acetic anhydride esterification product of camellia oleifera fruit shell.

[0012] Preferably, the ionic liquid is one of a mixed solution of dimethyl sulfoxide and 1-methylimidazole, a dimethyl sulfoxide solution of 1-butyl-3-methylimidazolium chloride, and a DMSO solution of LiCl.

[0013] Preferably, in the camellia oleifera shell solution, the addition amount ratio of acetic anhydride is: the amount of substance of acetic anhydride: the mass of camellia oleifera fruit shell = 40 - 45 mmol / g.

[0014] Preferably, the volume ratio of toluene to ethanol in the mixed solvent is 2 - 4:1.

[0015] The oil-tea camellia shell ester-based composite microspheres include the oil-tea camellia shell esterification product prepared by the preparation method of the oil-tea camellia shell esterification product described above, tea polyphenols, and poly(lactic-co-glycolic acid); the oil-tea camellia shell esterification product and poly(lactic-co-glycolic acid) are used as the skeleton carriers to embed the tea polyphenols in the microspheres.

[0016] Preferably, the mass ratio of the oil-tea camellia shell esterification product to poly(lactic-co-glycolic acid) is 1-3:1.

[0017] Preferably, the ratio of the tea polyphenols to the sum of the masses of the oil-tea camellia shell acetic anhydride esterification product and PLGA is 1:3-5.

[0018] The preparation method of the oil-tea camellia shell ester-based composite microspheres includes the following steps:

[0019] Using N,N-dimethylformamide and dichloromethane as the mixed solvent, adding the oil-tea camellia shell acetic anhydride esterification product and poly(lactic-co-glycolic acid), and continuously stirring at 45-55 °C until completely dissolved; adding tea polyphenols, fully stirring until completely dissolved, and standing for 15-20 min to obtain a uniform precursor solution;

[0020] Performing electrospray on the precursor solution and vacuum drying to obtain the oil-tea camellia shell ester-based composite microspheres.

[0021] The application of the oil-tea camellia shell ester-based composite microspheres is used for loading drugs.

[0022] Preferably, the drug is a drug for treating enteritis.

[0023] Preferably, the pulverizing and sieving is specifically pulverizing and sieving through a 100-120 mesh sieve.

[0024] Preferably, the addition amount of the ionic liquid is such that the mass concentration of the oil-tea camellia shell esterification product in the mixture obtained by the reaction is 8-10%.

[0025] Preferably, in the mixed solution of dimethyl sulfoxide and 1-methylimidazole, the volume ratio of dimethyl sulfoxide to 1-methylimidazole is 1.8-2.2:1.

[0026] Preferably, the concentration of the 1-butyl-3-methylimidazolium chloride dimethyl sulfoxide solution is 3-8%.

[0027] Preferably, the concentration of the LiCl DMSO solution is 5-12%.

[0028] Preferably, the electrospray is specifically:

[0029] Using a flat aluminum foil as the receiver, control the propulsion rate of the injection pump to be 0.5 - 1.5 mL / h, the distance between the receiver and the nozzle to be 10 - 20 cm, the voltage to be 12 - 20 kV, the inner diameter of the nozzle orifice to be 0.5 mm, the ambient temperature to be 25 - 35 °C, and the ambient humidity to be 50 - 60%.

[0030] Preferably, the drying temperature is 35 - 50 °C.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] (1) The preparation method of the esterified product of camellia fruit shell in the present invention performs homogeneous acetic anhydride esterification on the camellia fruit shell, which not only effectively improves the solubility of the camellia fruit shell, endows it with excellent processing performance, but also is conducive to the loading of drugs.

[0033] (2) The esterified product of camellia fruit shell in the present invention helps to improve the encapsulation efficiency of tea polyphenols and the antibacterial effect of microspheres.

[0034] (3) The ester-based composite microspheres of camellia fruit shell in the present invention have the characteristics of uniform particle size, high encapsulation efficiency and drug loading rate, good antibacterial and anti-inflammatory effects, good biocompatibility, and biodegradability.

[0035] (4) The ester-based composite microspheres of camellia fruit shell in the present invention can well encapsulate tea polyphenols, avoid the destruction of drugs by the acidic digestive environment, and promote the absorption of drugs by the intestinal mucosa.

[0036] (5) The ester-based composite microspheres of camellia fruit shell in the present invention can regulate the intestinal immune response and the intestinal microbiome, thereby treating intestinal inflammation.

[0037] (6) The ester-based composite microspheres of camellia fruit shell in the present invention have a certain colon-targeting effect, can deliver drugs to the inflammatory site, and improve the treatment effect.

[0038] (7) The raw materials used in the present invention are widely sourced, have good biocompatibility, and are biodegradable.

[0039] (8) The present invention realizes the high-value utilization of all components of the camellia fruit shell lignocellulose biomass, which is beneficial to enhancing the added value of the camellia industry. Description of the Drawings

[0040] Figure 1 It is the drug release curves of the ester-based composite microspheres of camellia fruit shell prepared in Example 5, Comparative Example 1, and Comparative Example 2 of the present invention in simulated artificial gastric juice.

[0041] Figure 2 It is the drug release curves of the ester-based composite microspheres of camellia fruit shell prepared in Example 5, Comparative Example 1, and Comparative Example 2 of the present invention in simulated artificial intestinal juice.

[0042] Figure 3 Drug release curves of the camellia oleifera shell ester-based composite microspheres prepared in Example 5, Comparative Example 1, and Comparative Example 2 of the present invention in simulated artificial colon fluid.

[0043] Figure 4 Drug release curve of the camellia oleifera shell ester-based composite microspheres prepared in Example 5 of the present invention in PBS buffer solution. Detailed implementation manners

[0044] The following further describes the present invention in detail in conjunction with embodiments, but the implementation manners of the present invention are not limited thereto.

[0045] Example 1

[0046] The camellia oleifera shell was crushed and sieved through a 110-mesh sieve, extracted in a toluene / ethanol (V / V = 2:1) mixed solvent at 80 °C for 5 h, and vacuum dried at 50 °C to constant weight. Take 0.5 g of camellia oleifera shell, add 4.5 g of dimethyl sulfoxide / 1-methylimidazole (V / V = 2:1) mixed solvent, stir at 100 °C for 6 h under nitrogen protection to completely dissolve the camellia oleifera shell. Then slowly add 20 mmol of acetic anhydride to the camellia oleifera shell solution and react at 100 °C for 90 min under nitrogen protection. Pour the resulting mixture into ethanol 5 - 10 times the volume of acetic anhydride to terminate the reaction, centrifuge and separate, and collect the solid after the reaction. The obtained product was vacuum dried at 50 °C to constant weight to obtain the camellia oleifera shell acetic anhydride esterification product.

[0047] Using N,N-dimethylformamide and dichloromethane as a mixed solvent (V / V = 3:1), add the camellia oleifera shell acetic anhydride esterification product and PLGA thereto (controlling the total mass concentration of the two to be 3%, and the mass ratio of the camellia oleifera shell acetic anhydride esterification product to PLGA to be 2:1), and continuously stir at 50 °C until completely dissolved. Then add tea polyphenols to the solution (controlling the mass ratio of tea polyphenols to the sum of the mass of the shell esterification product and PLGA to be 1:3), stir well until completely dissolved, and let stand for 15 min to obtain a uniform precursor solution.

[0048] Electrostatic spraying was carried out on the precursor solution, and vacuum drying was carried out at 40 °C to obtain the camellia oleifera shell ester-based composite microspheres. Electrostatic spraying parameters: the propulsion rate of the injection pump is 0.8 mL / h, the distance between the receiver and the nozzle is 10 cm, the voltage is 15 kV, the inner diameter of the nozzle orifice is 0.5 mm, the ambient temperature is 25 °C, and the ambient humidity is 60%.

[0049] Example 2

[0050] The oil-tea camellia fruit shell was crushed and passed through a 100-mesh sieve, extracted in a toluene / ethanol (V / V = 4:1) mixed solvent at 70 °C for 6 h, and vacuum dried at 50 °C to constant weight. Take 0.5 g of the oil-tea camellia fruit shell, add 4.5 g of a dimethyl sulfoxide solution of 5% 1-butyl-3-methylimidazolium chloride, stir at 110 °C for 4 h under nitrogen protection to completely dissolve the oil-tea camellia fruit shell. Then, slowly add 22.5 mmol of acetic anhydride to the oil-tea camellia fruit shell solution and react at 90 °C for 120 min under nitrogen protection. Pour the reaction mixture into ethanol 10 times the volume of acetic anhydride to terminate the reaction, centrifuge, and collect the solid after the reaction. The obtained product was vacuum dried at 50 °C to constant weight to obtain the oil-tea camellia fruit shell acetic anhydride esterification product.

[0051] Using N,N-dimethylformamide and dichloromethane as a mixed solvent (V / V = 4:1), add the oil-tea camellia fruit shell acetic anhydride esterification product and PLGA to it (control the total mass concentration of the two to be 4%, and the mass ratio of the oil-tea camellia fruit shell acetic anhydride esterification product to PLGA is 2:1), and continuously stir at 50 °C until completely dissolved. Then add tea polyphenols to the solution (control the mass ratio of tea polyphenols to the sum of the mass of the fruit shell esterification product and PLGA to be 1:5), stir well until completely dissolved, and let it stand for 15 min to obtain a uniform precursor solution.

[0052] Electrostatic spraying was carried out on the precursor solution, and vacuum drying was carried out at 40 °C to obtain the oil-tea camellia fruit shell ester-based composite microspheres. Electrostatic spraying parameters: the propulsion rate of the injection pump is 0.8 mL / h, the distance between the receiver and the nozzle is 10 cm, the voltage is 18 kV, the inner diameter of the nozzle is 0.5 mm, the ambient temperature is 25 °C, and the ambient humidity is 60%.

[0053] Example 3

[0054] The oil-tea camellia fruit shell was crushed and passed through a 120-mesh sieve, extracted in a toluene / ethanol (V / V = 3:1) mixed solvent at 90 °C for 4 h, and vacuum dried at 50 °C to constant weight. Take 0.5 g of the oil-tea camellia fruit shell, add 4.5 g of a DMSO solution of 8% LiCl, stir at 100 °C for 6 h under nitrogen protection to completely dissolve the oil-tea camellia fruit shell. Then, slowly add 21 mmol of acetic anhydride to the oil-tea camellia fruit shell solution and react at 100 °C for 100 min under nitrogen protection. Pour the reaction mixture into ethanol 8 times the volume of acetic anhydride to terminate the reaction, centrifuge, and collect the solid after the reaction. The obtained product was vacuum dried at 50 °C to constant weight to obtain the oil-tea camellia fruit shell acetic anhydride esterification product.

[0055] Using N,N-dimethylformamide and dichloromethane as a mixed solvent (V / V = 2:1), add the acetic anhydride esterification product of camellia oleifera fruit shell and PLGA (controlling the total mass concentration of the two to be 6%, and the mass ratio of the acetic anhydride esterification product of camellia oleifera fruit shell to PLGA is 1:1), and continuously stir at 50 °C until completely dissolved. Then add tea polyphenols to the solution (controlling the mass ratio of tea polyphenols to the sum of the mass of the fruit shell esterification product and PLGA to be 1:4), fully stir until completely dissolved, and let it stand for 15 min to obtain a uniform precursor solution.

[0056] Electrostatically spray the precursor solution and dry it in vacuum at 50 °C to obtain the camellia oleifera fruit shell ester-based composite microspheres. Electrostatic spraying parameters: the propulsion rate of the injection pump is 0.8 mL / h, the distance between the receiver and the nozzle is 15 cm, the voltage is 18 kV, the inner diameter of the nozzle orifice is 0.5 mm, the ambient temperature is 25 °C, and the ambient humidity is 60%.

[0057] Example 4

[0058] The difference between this example and Example 1 is that the total mass concentration of the acetic anhydride esterification product of camellia oleifera fruit shell and PLGA is 4%, the distance between the receiver and the nozzle is 15 cm, the voltage is 18 kV, and the others are the same as in Example 1.

[0059] Example 5

[0060] The difference between this example and Example 1 is that the total mass concentration of the acetic anhydride esterification product of camellia oleifera fruit shell and PLGA is 4%, the propulsion rate of the injection pump is 1.2 mL / h, the distance between the receiver and the nozzle is 15 cm, the voltage is 18 kV, and the others are the same as in Example 1.

[0061] Example 6

[0062] The difference between this example and Example 1 is that the total mass concentration of the acetic anhydride esterification product of camellia oleifera fruit shell and PLGA is 4%, the propulsion rate of the injection pump is 1.5 mL / h, the distance between the receiver and the nozzle is 20 cm, the voltage is 20 kV, and the others are the same as in Example 1.

[0063] Comparative Example 1

[0064] The difference between this comparative example and Example 5 is that the camellia oleifera fruit shell esterification product is not used, and the electrostatically sprayed precursor solution is a PLGA solution with a mass concentration of 3% containing an equal amount of tea polyphenols.

[0065] Comparative Example 2

[0066] A microemulsion system containing tea polyphenols was prepared in this comparative example, specifically as follows:

[0067] Linoleic acid was selected as the oil phase (and also as the surfactant), and ethanol was used as the co-surfactant to prepare a linoleic acid-water-ethanol reverse microemulsion containing tea polyphenols (where the mass ratio of the aqueous phase, oil phase, and co-surfactant was 1∶3∶4, and the mass concentration of tea polyphenols was 0.1 g / mL).

[0068] Test 1 Determination of the particle size, encapsulation efficiency, and drug loading of the oil-tea fruit shell ester-based composite microspheres prepared in Examples 1-6 and Comparative Examples 1-2

[0069] Method: Weigh 0.0100 g of the drug-loaded microspheres, add 2 mL of PBS solution, stir and mix well to form a sample solution, place it in a high-speed centrifuge, centrifuge at 15000 r / min for 10 min, filter the supernatant after centrifugation with a 0.22 μm filter membrane, measure the absorbance value of the filtrate at 540 nm, and calculate the encapsulation efficiency.

[0070] Weigh 0.0100 g of the drug-loaded microspheres, add 2 mL of anhydrous methanol and mix well. After ultrasonic treatment in an ultrasonic cell disruptor for 10 min, place it in a high-speed centrifuge, centrifuge at 15000 r / min for 20 min, filter the supernatant after centrifugation with a 0.22 μm filter membrane, measure the absorbance value of the filtrate at 540 nm, and calculate the drug loading.

[0071]

[0072]

[0073] In the formula, W p is the actual drug loading of the microspheres, W m is the amount of microspheres put in, and Q p is the initial amount of drug added to the microspheres.

[0074] The particle size of the microspheres was determined by a Malvern laser particle size analyzer, and the particle size of the microemulsion was determined by a Zetasizer Nano S nanometer particle size analyzer.

[0075] Results: The particle size, encapsulation efficiency, and drug loading of the oil-tea fruit shell ester-based composite microspheres prepared in Examples 1-6 are shown in Table 1. The particle size of the microspheres was 17-26 μm, the encapsulation efficiency was 80-92%, and the drug loading was 24-36%, indicating that the prepared microspheres can effectively achieve the encapsulation of tea polyphenols. The encapsulation efficiency and drug loading of Comparative Example 1 were lower than those of Examples 1-6, indicating that the acetic anhydride esterification product of the oil-tea fruit shell is helpful for drug loading. There was a small amount of drug loading in Comparative Example 2, indicating that the microemulsion has a certain encapsulation ability for tea polyphenols, but the encapsulation efficiency and drug loading were significantly lower than those of the microspheres.

[0076] Table 1 Average particle size, encapsulation efficiency, and drug loading of Examples 1-6 and Comparative Examples 1-2

[0077] Average particle size (μm) Entrapment efficiency (%) Drug loading (%) Example 1 22.21±0.17 85.46 28.88 Example 2 18.17±0.58 85.33 30.25 Example 3 19.45±0.42 87.18 32.76 Example 4 17.39±0.35 90.70 34.58 Example 5 20.84±0.18 92.41 36.53 Example 6 26.62±0.53 80.96 24.22 Comparative Example 1 19.33±0.66 63.52 16.34 Comparative Example 2 0.0100±0.0023 57.59 12.07

[0078] Stability Test of the Camellia oleifera Fruit Shell Ester-based Composite Microspheres Prepared in Example 5 of Test 2 and Comparative Examples 1 and 2 in Simulated Gastrointestinal Fluids

[0079] Method: Preparation of simulated gastric fluid (SGF): Dissolve 0.643 g of KCl, 2.625 g of NaHCO3, 0.153 g of KH2PO4, 0.031 g of MgCl2(H2O)6, 0.06 g of (NH4)2CO3 and 2.808 g of NaCl in 1 L of deionized water, and adjust the pH of the solution to 1.2 to obtain SGF. Add 10 g of pepsin to it to obtain E-SGF.

[0080] Preparation of simulated intestinal fluid (SIF): Dissolve 0.634 g of KCl, 8.952 g of NaHCO3, 0.136 g of KH2PO4, 0.084 g of MgCl2(H2O)6 and 3.452 g of NaCl in 1 L of deionized water, and adjust the pH of the solution to 6.8 to obtain SIF. Add 10 g of trypsin to it to obtain E-SIF.

[0081] Preparation of simulated colon fluid (SCF): Dissolve 8.5 g of NaCl, 0.13 g of NaH2PO4·2H2O and 2.2 g of anhydrous Na2HPO4 in 1 L of deionized water, and adjust the pH of the solution to 7.4. Add 10 g of lysozyme to it to obtain E-SCF.

[0082] In vitro simulated release of drugs in simulated gastric fluid: Weigh 0.1 g of the drug-loaded composite microspheres into 50 mL of E-SGF dissolution medium, with the medium temperature at 37 °C and the rotation speed at 100 r / min. According to the transit time of substances in the human gastrointestinal tract, set the release time in simulated gastric fluid to 2 h. Take 3 mL of the supernatant every 30 min, and then add 3 mL of dissolution medium. After centrifuging the taken supernatant, measure the absorbance at 540 nm and calculate the concentration, with 3 parallel determinations.

[0083] In vitro simulated release in simulated intestinal fluid: Weigh 0.1 g of the drug-loaded composite microspheres and place them in 50 mL of E-SIF dissolution medium, with the medium temperature at 37 °C and the rotation speed at 100 r / min. Set the release time in simulated intestinal fluid to 6 h. Take 3 mL of samples every 30 min from 0 to 3 h and every 1 h from 3 to 6 h, and then add 3 mL of dissolution medium. After centrifuging the taken supernatant, measure the absorbance at 540 nm and calculate the concentration, with 3 parallel determinations.

[0084] In vitro simulated release in artificial colon fluid: Weigh 0.1 g of the drug-loaded composite microspheres and place them in 50 mL of E-SCF dissolution medium. The medium temperature is 37 °C and the rotation speed is 100 r / min. The release time in artificial intestinal fluid is set to 30 h. Samples are taken every 30 min from 0 to 3 h, every 1 h from 3 to 7 h, and then every 5, 12, and 6 h in sequence. Each time, 3 mL of the supernatant is taken, and then 3 mL of dissolution medium is replenished into the sustained-release system. After centrifuging the taken supernatant, measure the absorbance at 540 nm and calculate the concentration, with 3 parallel determinations.

[0085] Results: The results show that within 2 h in gastric juice, the tea polyphenol emulsion of Comparative Example 2 releases drugs rapidly (81.9%), while the camellia oleifera shell ester-based composite microspheres prepared in Example 5 can release tea polyphenols slowly, with a cumulative release rate of only 12.4%; the cumulative release rate of Comparative Example 1 without using the esterification product of camellia oleifera shell is 20.3%, indicating that the microspheres prepared with the esterification product of camellia oleifera shell and PLGA as raw materials can effectively resist the destruction of gastric acid, and the esterification product of camellia oleifera shell helps to improve the stability of the microspheres in gastric juice.

[0086] In the small intestine digestion stage, it is observed that the tea polyphenol emulsion system prepared in Comparative Example 2 continuously releases a large amount of tea polyphenols in the first 2 h, but the camellia oleifera shell ester-based composite microspheres prepared in Example 5 continuously release drugs slowly, and the cumulative release rate is only 28.6% after 6 h of digestion in small intestinal fluid, which is significantly lower than that of the tea polyphenol emulsion of Comparative Example 2. This indicates that the prepared fruit shell ester-based composite microspheres containing tea polyphenols have high stability in simulated gastric and small intestinal digestion.

[0087] During the digestion process in colon fluid, the camellia oleifera shell ester-based composite microspheres continuously release tea polyphenols, with a cumulative release rate of 77.8%, while the cumulative release rate of the tea polyphenol emulsion of Comparative Example 2 is only 37.4%. The results show that the prepared fruit shell ester-based composite microspheres have colon-specific release characteristics.

[0088] Test 3 Detection of the tea polyphenol sustained-release performance of the camellia oleifera shell ester-based composite microspheres prepared in Example 5

[0089] Method: Precisely weigh 2 mg of the fruit shell ester-based composite microspheres, put them into a dialysis bag and suspend them in a centrifuge tube. Add 5 mL of PBS buffer solution, seal it, and place it in a shaker at a temperature of 37 °C and an oscillation frequency of 100 r / min for shaking. Take 1 mL of the release medium at certain time intervals, filter it through a microporous membrane, measure the absorbance at 540 nm, and at the same time replenish 1 mL of PBS buffer solution, and continue to shake under the same conditions. Repeat the operation steps. Use the standard curve of tea polyphenols to determine the release amount of tea polyphenols in the solution, and calculate the cumulative release rate of tea polyphenols of the composite microspheres. The time is set to 30 days.

[0090]

[0091] In the formula, M is the amount of tea polyphenols contained in the microspheres (μg); λi is the concentration of tea polyphenols in the centrifuge tube measured at the i-th time point (μg / mL); n is the number of tests.

[0092] Results: The results show that the oil-tea fruit shell ester-based composite microspheres prepared in Example 5 slowly release tea polyphenols within 0 - 30 days, the drug release is relatively complete, and it has good sustained-release performance.

[0093] Test 4 Determination of the antibacterial effects of the oil-tea fruit shell ester-based composite microspheres prepared in Examples 1 - 6 and Comparative Examples 1 - 2

[0094] Method: The antibacterial performance was determined by the shaking method. The strains selected for the antibacterial effect test were Staphylococcus aureus and Salmonella enteritidis, and the blank PBS solution was used as the control group. The antibacterial activity of the test samples was evaluated by the inhibition rate.

[0095] The calculation formula for the inhibition rate is

[0096]

[0097] In the formula, W is the average number of colonies in the culture dish of the standard blank sample, and Q is the average number of colonies in the culture dish of the drug-loaded microspheres with different drug dosages.

[0098] Results:

[0099] Table 2 Inhibition rates of Examples 1 - 6 and Comparative Examples 1 - 2 against Staphylococcus aureus and Salmonella enteritidis

[0100]

[0101] The inhibition rates were calculated to obtain Table 2. The data in the table show that the microspheres prepared in Examples 1 - 6 have good antibacterial effects against both Staphylococcus aureus and Salmonella enteritidis, and the inhibition rates against these two bacteria increase with the increase of the drug loading amount; the inhibition rates of the microspheres prepared in Comparative Example 1 against Staphylococcus aureus and Salmonella enteritidis are weaker than those in Example 1, indicating that the acetic anhydride esterification product of the oil-tea fruit shell has certain antibacterial effects; the antibacterial effect of the prepared fruit shell ester-based composite microspheres is much better than that of the tea polyphenol emulsion.

[0102] Test 5 Effects of the oil-tea fruit shell ester-based composite microspheres prepared in Example 5 on the contents of TNF-α, IL-1β, IL-6, IL-8 and IL-10 in rats

[0103] Method: Sixty healthy SPF-grade male SD rats weighing 180 - 200 g were randomly divided into 6 groups, including a blank control group (gavaged with normal saline), a model group (gavaged with 5% dextran sulfate sodium DSS solution, Mw: 45000 Da), a positive control group (gavaged with dexamethasone tablets), and experimental groups (low, medium, and high doses), with 10 rats in each group. They were randomly numbered and weighed. Before the experiment, the rats in each group were fasted for 12 h and allowed free access to water. The dosing amounts of the low, medium, and high doses in the experimental groups were 25 mg / kg, 50 mg / kg, and 100 mg / kg, respectively. Except for the blank control group, the other rats were gavaged with 5% DSS solution at 1.0 g / kg once a day for 7 consecutive days to establish an enteritis rat model. Except for the blank control group and the model group, starting from the 4th day of enteritis rat model establishment, different doses of the therapeutic drug were gavaged once a day for 7 consecutive days. On the 1st, 4th, and 7th days of dosing, 0.5 mL of blood was collected from the tail vein of the rats, allowed to stand for 2 h, and then centrifuged at low temperature (4°C, 3500 r / min, 20 min) to separate the serum. The complement was inactivated in a water bath at 56°C for 30 min, filtered through a 0.22 μm filter membrane, sealed and aliquoted, and stored at -20°C for later use. Commercial kits were used to detect the contents of indicators such as tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10) in the rats. All detections were carried out according to the kit instructions provided by the manufacturer.

[0104] Results: The experimental results are shown in Table 3 - 7.

[0105] Table 3 Effects of the present invention on TNF-α in enteritis rats (x±s, n = 10)

[0106]

[0107] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05,

[0108] (4) P < 0.01.

[0109] Table 4 Effects of the present invention on IL-1β in enteritis rats (x±s, n = 10)

[0110]

[0111] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05, (4)P < 0.01.

[0112] Table 5 Effects of the present invention on IL-6 in rats with enteritis (x±s, n = 10)

[0113]

[0114] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05, (4) P < 0.01.

[0115] Table 6 Effects of the present invention on IL-8 in rats with enteritis (x±s, n = 10)

[0116]

[0117] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05,

[0118] (4) P < 0.01.

[0119] Table 7 Effects of the present invention on IL-10 in rats with enteritis (x±s, n = 10)

[0120]

[0121] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05,

[0122] (4) P < 0.01.

[0123] The experimental group had varying degrees of effects on the inflammatory cytokines in the serum of rats with enteritis. Compared with the blank control group, the contents of TNF-ɑ, IL-1β, IL-6, and IL-8 in the serum of rats in the model group were significantly increased (P < 0.01), while the content of IL-10 was significantly decreased (P < 0.01); compared with the model group, after treatment with different doses of drugs in the experimental group, the medium-dose group and high-dose group could significantly inhibit the increase in the contents of TNF-ɑ, IL-1β, IL-6, and IL-8 and the decrease in the content of IL-10, and there were significant differences between groups (P < 0.05, P < 0.01), and there was no significant difference between the experimental groups (P > 0.05).

[0124] Effect of the Camellia oleifera fruit shell ester-based composite microspheres prepared in Example 5 of Test 6 on the contents of DAO, GSH-PX, MPO and MDA in rat colon homogenate

[0125] Method: On the 7th day of administration, the rats in each group were anesthetized and sacrificed. Immediately, 2 cm of colon tissue 6 - 8 cm away from the anus was intercepted, washed with normal saline at 4°C, and the surface moisture was blotted dry with filter paper, and then accurately weighed. It was ground with normal saline at a mass ratio of 1:9, and the grinding liquid was centrifuged at 3000 - 4000 r / min for 15 min. The supernatant was taken to prepare 10% tissue homogenate. The contents of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH-PX), and diamine oxidase (DAO) in rats were detected using an ELISA kit, and the operation was carried out according to the kit instructions.

[0126] Results:

[0127] Table 8 Effect of the present invention on MDA, MPO, GSH-PX, and DAO in rats with enteritis (x±s, n = 10)

[0128]

[0129] Note: Compared with the blank control group, (1) P < 0.05, (2) P < 0.01; compared with the model group, (3) P < 0.05,

[0130] (4) P < 0.01.

[0131] The experimental results are shown in Table 8. Compared with the blank control group, the contents of MDA and MPO in the colon tissue of the model group rats were significantly increased (P < 0.01), while the contents of GSH-PX and DAO were significantly decreased (P < 0.01). Compared with the model group, after treatment with different doses of drugs in the experimental group, the medium-dose and high-dose groups in the experimental group could significantly inhibit the increase in the contents of MDA and MPO and the decrease in the contents of GSH-PX and DAO, and there were significant differences between groups (P < 0.05, P < 0.01), and there was no significant difference between the experimental groups (P > 0.05).

[0132] Test 7 In vitro cytotoxicity evaluation of the Camellia oleifera fruit shell ester-based composite microspheres prepared in Examples 1 - 6 and Comparative Examples 1 - 2

[0133] The samples prepared in Examples 1 - 6 and Comparative Examples 1 - 2 were taken and evaluated and scored according to the requirements of GB / T 16886.5. The experimental results are as follows in the table, indicating that each example has no cytotoxicity.

[0134] Table 9 In vitro cytotoxicity scores of the samples prepared in Examples 1 - 6 and Comparative Examples 1 - 2

[0135]

[0136]

[0137] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the said embodiments. Any other changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing an esterification product of camellia oleifera fruit shell, characterized in that, It includes the following steps: Crush and sieve the camellia oleifera fruit shell, extract it in a mixed solvent of toluene and ethanol at 70 - 90 °C for 4 - 6 h, dry the residue under vacuum until constant weight, add an ionic liquid, stir at 100 - 110 °C for 4 - 6 h under nitrogen protection to completely dissolve the camellia oleifera shell, obtaining a camellia oleifera shell solution; then add acetic anhydride, react at 90 - 100 °C for 90 - 120 min under nitrogen protection, pour the reaction mixture into ethanol to terminate the reaction, centrifuge and separate, collect the solid after the reaction, and dry it under vacuum until constant weight to obtain the acetic anhydride esterification product of camellia oleifera fruit shell; Among them, the ionic liquid is one of a mixed solution of dimethyl sulfoxide and 1 - methylimidazole, a dimethyl sulfoxide solution of 1 - butyl - 3 - methylimidazolium chloride, and a DMSO solution of LiCl; In the camellia oleifera shell solution, the addition amount ratio of acetic anhydride is: the amount of substance of acetic anhydride: the mass of camellia oleifera fruit shell = 40 - 45 mmol / g.

2. The preparation method of the esterification product of camellia oleifera fruit shell according to claim 1, characterized in that, In the mixed solvent, the volume ratio of toluene to ethanol is 2 - 4:

1.

3. The ester-based composite microspheres of oil-tea fruit shell, characterized in that It includes the acetic anhydride esterification product of camellia oleifera fruit shell prepared by the preparation method of the acetic anhydride esterification product of camellia oleifera fruit shell described in any one of claims 1 - 2, tea polyphenols, and poly(lactic - co - glycolic acid); the acetic anhydride esterification product of camellia oleifera fruit shell and poly(lactic - co - glycolic acid) are used as a skeleton carrier to embed tea polyphenols in microspheres; the mass ratio of the acetic anhydride esterification product of camellia oleifera fruit shell to poly(lactic - co - glycolic acid) is 1 - 3:

1.

4. The ester-based composite microspheres of camellia oleifera fruit shell according to claim 3, characterized in that, The ratio of tea polyphenols to the sum of the masses of the acetic anhydride esterification product of camellia oleifera fruit shell and PLGA is 1:3 - 5.

5. The preparation method of the camellia oleifera fruit shell ester-based composite microspheres according to any one of claims 3 to 4, characterized in that, It includes the following steps: Using N,N - dimethylformamide and dichloromethane as a mixed solvent, add the acetic anhydride esterification product of camellia oleifera fruit shell and poly(lactic - co - glycolic acid), and continuously stir at 45 - 55 °C until completely dissolved; add tea polyphenols, fully stir until completely dissolved, and let it stand for 15 - 20 min to obtain a uniform precursor solution; Perform electrospray on the precursor solution and dry it under vacuum to obtain the camellia oleifera fruit shell ester - based composite microspheres.

6. Use of the oil-tea fruit shell ester-based composite microspheres according to any one of claims 3 to 4, characterized in that It is used for preparing a drug for treating intestinal inflammation.

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