A protein / polysaccharide nanosphere with intestinal targeting delivery function and its application

Abstract

The present invention belongs to the technical field of drug carriers, and particularly relates to a protein / polysaccharide nanosphere with intestinal targeting delivery function and its application. The protein / polysaccharide nanosphere with intestinal targeting delivery function provided by the present invention is prepared from zein, composite polysaccharide and calcium salt as main raw materials. The protein / polysaccharide nanosphere with intestinal targeting delivery function provided by the present invention has a high drug loading rate, excellent water dispersibility, and a good protective effect on the stability of drugs and bioactive components. It releases less in the gastric digestion stage and mainly releases in the intestinal digestion stage, having an intestinal targeting release function, and is a natural biomacromolecule nanocarrier.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and particularly relates to a protein / polysaccharide nanosphere with intestinal targeting delivery function and its application. Background Art

[0002] Some drugs or bioactive substances have low polarity, are insoluble in water but soluble in ethanol, and are sensitive to environmental pressures such as light, heat, and pH, etc., thus resulting in low bioavailability and biological activity, and affecting the activity of drugs and bioactive substances. Loading these drugs and bioactive components into microcarriers can increase their stability and water dispersibility. These carriers include solid nano-lipids, liposomes, microemulsions, nanoemulsions, protein nanoparticles, etc.

[0003] Zein is a storage protein derived from corn seeds, which has high hydrophobicity, is insoluble in water but soluble in high-concentration ethanol. Drugs or bioactive components and zein are co-dissolved in ethanol and then dispersed in water to form nanoparticles loaded with the target components. Chinese Patent Application CN107595809A discloses a zein nano-embedded sustained-release filler and its preparation method. The zein nano-embedded sustained-release filler is prepared from the following raw materials in parts by weight: 5 - 15 parts of zein, and 2 - 10 parts of an anti-tumor drug. The preparation method includes adding zein to an ethanol aqueous solution and stirring until the solution is uniformly transparent; adding an anti-tumor drug to an ethanol aqueous solution or water and stirring until the solution is uniformly transparent; mixing the anti-tumor drug ethanol aqueous solution or the anti-tumor drug aqueous solution with the zein solution; and subjecting the zein nano-embedded mixture to freeze-drying or spray-drying to obtain the zein nano-embedded sustained-release filler. However, zein has high hydrophobicity, low stability in water, is prone to aggregation, and the dried particles cannot be redispersed in water. Moreover, zein is rapidly disintegrated by pepsin in the stomach and cannot effectively protect the stability of the target components in a highly acidic environment. Therefore, it is necessary to develop a natural biopolymer nanocarrier with good water solubility and capable of releasing drugs in the intestinal digestion stage for the intestinal targeting delivery of drugs and bioactive components. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a protein / polysaccharide nanosphere with intestinal targeting delivery function. The protein / polysaccharide nanosphere with intestinal targeting delivery function provided by the present invention has a high drug loading rate, a particle size range of 100 - 500 nm, excellent water dispersibility, has a good protective effect on the stability of drugs and bioactive components, has a low release in the gastric digestion stage, mainly releases in the intestinal digestion stage, has an intestinal targeting release function, and is a natural biopolymer nanocarrier.

[0005] The technical solution of the present invention is as follows:

[0006] A protein / polysaccharide nanosphere with intestinal targeting delivery function, and the protein / polysaccharide nanosphere is prepared from zein, composite polysaccharide and calcium salt as main raw materials.

[0007] Furthermore, the composite polysaccharide is two or three of gum arabic, pectin, sodium alginate, carrageenan, and propylene glycol alginate.

[0008] Furthermore, the composite polysaccharide is composed of gum arabic / carrageenan / propylene glycol alginate, pectin and sodium alginate in a mass ratio of 0.1-5:65-75:20-34.9.

[0009] Furthermore, the composite polysaccharide is composed of propylene glycol alginate, pectin and sodium alginate in a mass ratio of 5:75:20; or the composite polysaccharide is composed of carrageenan, pectin and sodium alginate in a mass ratio of 0.1:75:24.9; or the composite polysaccharide is composed of gum arabic, pectin and sodium alginate in a mass ratio of 5:72.5:22.5.

[0010] Furthermore, the calcium salt is calcium chloride or calcium lactate.

[0011] The present invention also provides a preparation method of the protein / polysaccharide nanosphere with intestinal targeting delivery function, including the following steps:

[0012] S1. Dissolve zein in an ethanol aqueous solution with a volume fraction of 60-90%, so that the concentration of zein is 1-3% (w / v) to obtain an ethanol solution of zein;

[0013] S2. Dissolve the composite polysaccharide in water, so that the concentration of the composite polysaccharide is 0.1-0.5% (w / v), and adjust the pH to 4.0-7.0 to obtain a polysaccharide solution;

[0014] S3. Disperse the ethanol solution of zein obtained in step S1 into the polysaccharide solution obtained in step S2, and the volume ratio of the ethanol solution of zein to the polysaccharide solution is 1:2-1:10, and disperse by high-speed stirring for 3-8 minutes to obtain a particle dispersion;

[0015] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion;

[0016] S5. Add calcium salt to the dispersion obtained in step S4 so that the final concentration of calcium ions is 1-50 mM, and stir for 7-15 minutes;

[0017] S6. Freeze-dry or spray-dry to obtain the product.

[0018] Further, when performing high-speed stirring and dispersion in step S3, the stirring speed is 800 - 1200 revolutions per minute.

[0019] Another object of the present invention is to provide the application of the protein / polysaccharide nanospheres with intestinal targeting delivery function in the preparation of an intestinal targeting carrier loaded with drugs or active ingredients.

[0020] The present invention uses an anti-solvent method combined with an electrostatic deposition method to prepare zein as the core (loaded with drugs or bioactive substances). The isoelectric point of zein is pH 6.2. When the pH is lower than the isoelectric point, the zein nanoparticles prepared by the anti-solvent method have electrostatic interactions with anionic composite polysaccharides, and the composite polysaccharides are adsorbed onto the surface of the nanoparticles to form core / shell composite nanoparticles. The strength of the composite polysaccharide shell layer is adjusted by the electrostatic interaction between calcium ions and anionic groups of the polysaccharides such as carboxyl or sulfate groups, thereby protecting the zein nanoparticles from being decomposed by gastric acid and pepsin and releasing the loaded drugs and active ingredients. Under the neutral pH conditions in the intestine, the solubility of the polysaccharides in water increases, and the zein nanoparticles are hydrolyzed by trypsin to release the loaded drugs and bioactive substances, thus achieving intestinal targeted release.

[0021] Since zein is a hydrophobic protein, the prepared nanoparticles are prone to aggregation in water, and the dried nanoparticles are difficult to disperse in water. In the present invention, a hydrophilic composite polysaccharide forms an electrostatic composite shell layer on the surface of the nanoparticles, and calcium ions strengthen the polysaccharide colloid shell layer. The hydrophilic composite polysaccharide covers the surface of the zein nanoparticles, increasing the polarity of the particles, so that the prepared nanoparticles can be dispersed in water.

[0022] The key technology of the present invention is to optimize the type and proportion of polysaccharides, the type and concentration of calcium ions to regulate the hydrophilicity of the polysaccharide shell layer, the dispersibility of the particles in different pH water environments, and the intestinal targeting release characteristics.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The raw materials used in the present invention are natural edible proteins, polysaccharides and edible calcium salts. No chemical cross-linking agents are used in the production and processing process, which are non-toxic, harmless and have good biocompatibility.

[0025] 2. The nanospheres prepared by the present invention are uniform in size, the particle size does not exceed 500 nm, the drug loading rate is high, and they have excellent water dispersibility.

[0026] 3. The nanospheres prepared by the present invention have a good protective effect on the stability of drugs and bioactive ingredients. They have a low release rate during the gastric digestion stage and mainly release during the intestinal digestion stage, and have an intestinal targeting release function.

[0027] 4. The protein / polysaccharide nanospheres with intestinal targeting delivery function of the present invention have broad application prospects in the fields such as carriers for loading drugs and active ingredients, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following is a detailed description of this experiment in combination with the drawings and specific embodiments:

[0029] Figure 1 Appearance of the nanoparticles obtained in step S5 of Example 5 of the present invention;

[0030] Figure 2 Appearance of the nanoparticles obtained in step S5 of Comparative Example 2;

[0031] Figure 3 Appearance of the nanoparticles obtained in step S5 of Comparative Example 3;

[0032] Figure 4 Scanning electron microscope image of zein / composite polysaccharide nanospheres loaded with quercetin prepared in Example 4 of the present invention;

[0033] Figure 5 Comparison diagram of the redispersibility of the nanospheres;

[0034] Figure 6 Change diagram of the release rate of quercetin in simulated gastric juice at different digestion times;

[0035] Figure 7 Change diagram of the release rate of quercetin in simulated intestinal fluid at different digestion times. SPECIFIC EMBODIMENTS

[0036] The present invention will be further described below through the description of specific embodiments. However, this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention. As long as they do not depart from the basic idea of the present invention, they are within the scope of the present invention.

[0037] The raw materials used in the present invention are all commercially available unless otherwise specified.

[0038] Example 1. A zein / polysaccharide nanosphere with intestinal targeting delivery function and its preparation method

[0039] The zein / polysaccharide nanosphere with intestinal targeting delivery function is prepared from zein, composite polysaccharide and calcium salt as main raw materials. The composite polysaccharide is composed of carrageenan, pectin and sodium alginate according to the mass ratio of 0.1:75:24.9, and the calcium salt is calcium chloride.

[0040] The preparation method of the zein / polysaccharide nanosphere with intestinal targeting delivery function includes the following steps:

[0041] S1. Dissolve zein in an ethanol aqueous solution with a volume fraction of 80%, so that the concentration of zein is 1% (w / v) to obtain an ethanol solution of zein;

[0042] S2. Dissolve the complex polysaccharide in water, so that the concentration of the complex polysaccharide is 0.1% (w / v), and adjust the pH to 4.2 to obtain a polysaccharide solution;

[0043] S3. Rapidly inject and disperse the ethanol solution of zein obtained in step S1 into the polysaccharide solution obtained in step S2 with a syringe. The volume ratio of the ethanol solution of zein to the polysaccharide solution is 1:2, and disperse it by high-speed stirring at a stirring speed of 1000 revolutions per minute for 3 minutes to obtain a particle dispersion;

[0044] S4. Rotate and evaporate the ethanol in the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion;

[0045] S5. Add a calcium salt to the dispersion obtained in step S4 so that the final concentration of calcium ions is 5 mM, and stir for 7 minutes;

[0046] S6. Freeze-dry to obtain the product.

[0047] Example 2. A zein / polysaccharide nanosphere with intestinal targeting delivery function and its preparation method

[0048] The zein / polysaccharide nanosphere with intestinal targeting delivery function is prepared from zein, complex polysaccharide and calcium salt as main raw materials. The complex polysaccharide is composed of propylene glycol alginate, low-ester pectin and sodium alginate according to a mass ratio of 0.5:70:29.5, and the calcium salt is calcium lactate.

[0049] The preparation method of the zein / polysaccharide nanosphere with intestinal targeting delivery function includes the following steps:

[0050] S1. Dissolve zein in an ethanol aqueous solution with a volume fraction of 85%, so that the concentration of zein is 3% (w / v) to obtain an ethanol solution of zein;

[0051] S2. Dissolve the complex polysaccharide in water, so that the concentration of the complex polysaccharide is 0.5% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution;

[0052] S3. Rapidly inject and disperse the ethanol solution of zein obtained in step S1 into the polysaccharide solution obtained in step S2 with a syringe. The volume ratio of the ethanol solution of zein to the polysaccharide solution is 1:5, and disperse it by high-speed stirring at a stirring speed of 1200 revolutions per minute for 8 minutes to obtain a particle dispersion;

[0053] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion.

[0054] S5. Add calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ions 9 mM, and stir for 15 minutes.

[0055] S6. Freeze-dry to obtain the product.

[0056] Example 3. A zein / polysaccharide nanosphere with intestinal targeting delivery function and its preparation method

[0057] The zein / polysaccharide nanosphere with intestinal targeting delivery function is prepared from zein, composite polysaccharide and calcium salt as main raw materials. The composite polysaccharide is composed of pectin, sodium alginate and arabic gum according to the mass ratio of 72.5:22.5:5, and the calcium salt is calcium chloride.

[0058] The preparation method of the zein / polysaccharide nanosphere with intestinal targeting delivery function includes the following steps:

[0059] S1. Dissolve zein in an ethanol aqueous solution with a volume fraction of 80% to make the concentration of zein 2% (w / v) to obtain an ethanol solution of zein.

[0060] S2. Dissolve the composite polysaccharide in water to make the concentration of the composite polysaccharide 0.1% (w / v), and adjust the pH to 4.2 to obtain a polysaccharide solution.

[0061] S3. Quickly disperse the ethanol solution of zein obtained in step S1 into the polysaccharide solution obtained in step S2 with a syringe. The volume ratio of the ethanol solution of zein to the polysaccharide solution is 1:4, and disperse by high-speed stirring. The stirring speed is 1000 revolutions per minute, and the stirring time is 5 minutes to obtain a particle dispersion.

[0062] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion.

[0063] S5. Add calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ions 5 mM, and stir for 10 minutes.

[0064] S6. Freeze-dry to obtain the product.

[0065] Example 4. A quercetin-loaded zein / composite polysaccharide nanosphere and its preparation method

[0066] The quercetin-loaded zein / composite polysaccharide nanospheres are prepared from zein, quercetin, composite polysaccharide and calcium salt as main raw materials. The composite polysaccharide is composed of sodium alginate, pectin and propylene glycol alginate according to a mass ratio of 20:75:5, and the calcium salt is calcium chloride.

[0067] The preparation method of the quercetin-loaded zein / composite polysaccharide nanospheres comprises the following steps:

[0068] S1. Dissolve zein and quercetin in an ethanol aqueous solution with a volume fraction of 80% to make the concentration of zein 2% (w / v) and the concentration of quercetin 0.3% (w / v) to obtain a mixed solution.

[0069] S2. Dissolve the composite polysaccharide in water to make the concentration of the composite polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution.

[0070] S3. Disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 by means of injection with a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5, and disperse it by high-speed stirring. The stirring speed is 1000 revolutions per minute, and the stirring time is 5 minutes to obtain a particle dispersion.

[0071] S4. Rotate and evaporate the ethanol in the particle dispersion obtained in step S3, and add water to compensate for the volume of the evaporated ethanol to obtain a dispersion.

[0072] S5. Add calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ion 9 mM, and stir for 10 minutes.

[0073] S6. Freeze-dry to obtain the product.

[0074] Example 5. A quercetin-loaded zein / composite polysaccharide nanosphere and its preparation method

[0075] The quercetin-loaded zein / composite polysaccharide nanospheres are prepared from zein, quercetin, composite polysaccharide and calcium salt as main raw materials. The composite polysaccharide is composed of sodium alginate, pectin and propylene glycol alginate according to a mass ratio of 20:75:5, and the calcium salt is calcium chloride.

[0076] The preparation method of the quercetin-loaded zein / composite polysaccharide nanospheres comprises the following steps:

[0077] S1. Dissolve zein and quercetin in an ethanol aqueous solution with a volume fraction of 80% to make the concentration of zein 2% (w / v) and the concentration of quercetin 0.3% (w / v) to obtain a mixed solution.

[0078] S2. Dissolve the complex polysaccharide in water to make the concentration of the complex polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution.

[0079] S3. Disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 by injection with a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5. Stir and disperse at a high speed. The stirring speed is 1000 revolutions per minute, and the stirring time is 5 minutes to obtain a particle dispersion.

[0080] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion.

[0081] S5. Add a calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ions 5 mM, and stir for 10 minutes. The appearance of the nanoparticles obtained in step S5 of Example 5 of the present invention is as Figure 1 shown. It can be seen from Figure 1 this that a stable particle suspension is formed in this example.

[0082] S6. Freeze-dry to obtain the product.

[0083] Example 6. A zein / complex polysaccharide nanomicelle loaded with resveratrol and curcumin and its preparation method

[0084] The zein / complex polysaccharide nanomicelle loaded with resveratrol and curcumin is prepared from zein, resveratrol, curcumin, complex polysaccharide and calcium salt as main raw materials. The complex polysaccharide is composed of carrageenan, pectin and sodium alginate according to a mass ratio of 0.1:75:24.9. The calcium salt is calcium lactate.

[0085] The preparation method of the zein / complex polysaccharide nanomicelle loaded with resveratrol and curcumin includes the following steps:

[0086] S1. Stir and disperse resveratrol and curcumin in an ethanol aqueous solution with a volume fraction of 80%. This process should be carried out in the dark, and then add zein and continue to stir to dissolve it fully. Make the concentration of zein 2% (w / v), the concentration of resveratrol 0.5% (w / v), and the concentration of curcumin 0.3% (w / v) to obtain a mixed solution.

[0087] S2. Dissolve the complex polysaccharide in water to make the concentration of the complex polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution.

[0088] S3. Rapidly inject and disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 using a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5. Disperse by high-speed stirring at a stirring speed of 1200 revolutions per minute for 5 minutes to obtain a particle dispersion;

[0089] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion;

[0090] S5. Add calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ions 5 mM, and stir for 10 minutes;

[0091] S6. Freeze-dry to obtain the product.

[0092] Comparative Example 1. A zein / composite polysaccharide nanosphere loaded with quercetin

[0093] The zein / composite polysaccharide nanosphere loaded with quercetin is prepared from zein, quercetin, and composite polysaccharide as the main raw materials. The composite polysaccharide is composed of sodium alginate, pectin, and propylene glycol alginate in a mass ratio of 20:75:5.

[0094] The preparation method of the zein / composite polysaccharide nanosphere loaded with quercetin includes the following steps:

[0095] S1. Dissolve zein and quercetin in an ethanol aqueous solution with a volume fraction of 80% to make the concentration of zein 2% (w / v) and the concentration of quercetin 0.3% (w / v) to obtain a mixed solution;

[0096] S2. Dissolve the composite polysaccharide in water to make the concentration of the composite polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution;

[0097] S3. Rapidly inject and disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 using a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5. Disperse by high-speed stirring at a stirring speed of 1000 revolutions per minute for 5 minutes to obtain a particle dispersion;

[0098] S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion;

[0099] S5. Freeze-dry to obtain the product.

[0100] Comparative Example 2. A zein / composite polysaccharide nanosphere loaded with quercetin

[0101] The quercetin-loaded zein / composite polysaccharide nanoparticles are prepared from zein, quercetin, and composite polysaccharide as the main raw materials. The composite polysaccharide is composed of sodium alginate, pectin, and propylene glycol alginate in a mass ratio of 75:20:5, and the calcium salt is calcium chloride.

[0102] The preparation method of the quercetin-loaded zein / composite polysaccharide nanoparticles comprises the following steps:

[0103] S1. Dissolve zein and quercetin in an ethanol aqueous solution with a volume fraction of 80% to make the concentration of zein 2% (w / v) and the concentration of quercetin 0.3% (w / v) to obtain a mixed solution.

[0104] S2. Dissolve the composite polysaccharide in water to make the concentration of the composite polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution.

[0105] S3. Rapidly inject and disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 with a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5, and disperse it by high-speed stirring at a stirring speed of 1000 revolutions per minute for 5 minutes to obtain a particle dispersion.

[0106] S4. Rotate and evaporate the ethanol in the particle dispersion obtained in step S3, and add water to compensate for the volume of the evaporated ethanol to obtain a dispersion.

[0107] S5. Add the calcium salt to the dispersion obtained in step S4 to make the final concentration of calcium ions 5 mM, and stir for 10 minutes.

[0108] The appearance of the nanoparticles obtained in step S5 of Comparative Example 2 is as Figure 2 shown. It can be Figure 2 seen that the nanoparticles obtained in this comparative example are aggregated and precipitated, and stable nanoparticles cannot be prepared, indicating the regulating effect of the composition of the composite polysaccharide in the present invention on the nanoparticles.

[0109] Comparative Example 3. A quercetin-loaded zein / composite polysaccharide nanoparticle

[0110] The quercetin-loaded zein / composite polysaccharide nanoparticles are prepared from zein, quercetin, and composite polysaccharide as the main raw materials. The composite polysaccharide is composed of sodium alginate and pectin in a mass ratio of 75:25, and the calcium salt is calcium chloride.

[0111] The preparation method of the quercetin-loaded zein / composite polysaccharide nanoparticles comprises the following steps:

[0112] S1. Dissolve zein and quercetin in an ethanol aqueous solution with a volume fraction of 80%, so that the concentration of zein is 2% (w / v) and the concentration of quercetin is 0.3% (w / v) to obtain a mixed solution;

[0113] S2. Dissolve the composite polysaccharide in water to make the concentration of the composite polysaccharide 0.2% (w / v), and adjust the pH to 4.0 to obtain a polysaccharide solution;

[0114] S3. Quickly inject and disperse the mixed solution obtained in step S1 into the polysaccharide solution obtained in step S2 with a syringe. The volume ratio of the mixed solution to the polysaccharide solution is 1:5, and disperse it by high-speed stirring. The stirring speed is 1000 revolutions per minute and the stirring time is 5 minutes to obtain a particle dispersion;

[0115] S4. Rotate and evaporate the ethanol in the particle dispersion obtained in step S3, and add water to compensate for the volume of the evaporated ethanol to obtain a dispersion;

[0116] S5. Add a calcium salt to the dispersion obtained in step S4 so that the final concentration of calcium ions is 5 mM, and stir for 10 minutes;

[0117] The appearance of the nanoparticles obtained in step S5 of Comparative Example 3 is as Figure 3 shown. It can be seen from Figure 3 that the nanoparticles obtained in this comparative example aggregated and precipitated, and stable nanoparticles could not be prepared, indicating the regulatory effect of the composition of the composite polysaccharide on the nanoparticles in the present invention.

[0118] Comparative Example 4. Physical mixture of quercetin / zein / composite polysaccharide

[0119] A mixture obtained by physically mixing quercetin, zein, and composite polysaccharide in Example 5. Experimental Example 1. Scanning electron microscopy analysis of nanospheres

[0120] The morphology of the zein / composite polysaccharide nanospheres loaded with quercetin prepared in Example 4 was characterized by a scanning electron microscope, as shown in Figure 4 . It can be seen from Figure 4 that the nanospheres prepared in the present invention are uniform in size, and the particle size does not exceed 500 nm.

[0121] Experimental Example 2. Redispersibility of nanospheres

[0122] Take the dispersions obtained in Step S5 of Example 4 and Example 5 freshly prepared, and the dispersion obtained in Step S4 of Comparative Example 1, and measure the particle sizes of the above three kinds of nanoparticles respectively, which are recorded as the particle sizes of the freshly prepared nanoparticles. Disperse the freeze-dried nanoparticles prepared in Example 4, Example 5, and Comparative Example 1 into distilled water at a concentration of 5 mg / mL respectively, stir for 2 hours to obtain the re-dispersed nanoparticles, and measure the particle size of the re-dispersed nanoparticles (before re-dispersion heating). Place the re-dispersed nanoparticles in an 80°C water bath, heat and stir for 30 minutes, and measure the particle size of the nanoparticles after re-dispersion heating (after re-dispersion heating). And compare the particle sizes of the nanoparticles before and after re-dispersion heating with the particle sizes of the freshly prepared nanoparticles. The experimental results are as Figure 5 shown. It can be seen from Figure 5 that the particle size of the nanoparticles with calcium ions added after re-dispersion is close to that of the freshly prepared nanoparticles, indicating that calcium ions can regulate the re-dispersibility of the dried nanoparticles.

[0123] Experimental Example III: Determination of the drug loading amount and loading rate of the nanospheres

[0124] (I) Determine the drug loading amount and loading rate of the zein / composite polysaccharide nanospheres loaded with quercetin obtained in Example 4.

[0125] The specific steps are as follows:

[0126] Dissolve 1 mg of the freeze-dried zein / composite polysaccharide nanospheres loaded with quercetin obtained in Example 4 in 10 mL of DMSO, stir in the dark for 20 min, and then measure the absorbance value at a wavelength of 378 nm using a UV spectrophotometer. Establish a quercetin standard curve using a quercetin standard solution in the range of 0 - 10 μg / mL. The mass fraction of quercetin in the nanoparticles can be calculated through the standard curve, and thus the mass of quercetin in the nanoparticles can be deduced.

[0127] The drug loading amount of the nanoparticles and the quercetin loading rate are calculated respectively by the following formulas:

[0128]

[0129]

[0130] The measurement results show that the drug loading amount of the zein / composite polysaccharide nanospheres loaded with quercetin obtained in Example 4 of the present invention is 8.4%, and the loading rate is 89.1%.

[0131] (II) Determine the drug loading amount and loading rate of the zein / composite polysaccharide nanospheres loaded with resveratrol and curcumin obtained in Example 6.

[0132] The specific steps are as follows:

[0133] 1 mg of the zein / composite polysaccharide nanoparticles loaded with resveratrol and curcumin obtained in Example 6 of freeze-drying was dissolved in 10 mL of DMSO. After stirring for 20 min in the dark, the absorbance values were measured by an ultraviolet-visible spectrophotometer at wavelengths of 327 nm (resveratrol) and 435 nm (curcumin). The mass fractions of resveratrol and curcumin in the nanoparticles could be calculated through the standard curve. The drug loading and encapsulation efficiency of the nanoparticles were calculated by the following formulas respectively:

[0134]

[0135]

[0136] The measurement results showed that the drug loadings of the zein / composite polysaccharide nanoparticles loaded with quercetin obtained in Example 6 of the present invention for resveratrol and curcumin were 8.1% and 3.7% respectively, and the encapsulation efficiencies were 88.3% and 92.5% respectively. Thus, it can be seen that the drug encapsulation efficiency of the nanoparticles of the present invention is high.

[0137] Test Example 4: Release behavior of quercetin-loaded nanoparticles during simulated gastrointestinal digestion

[0138] The zein / composite polysaccharide nanoparticles loaded with quercetin prepared in Example 4 (quercetin nanoparticles + calcium 9 mM), the zein / composite polysaccharide nanoparticles loaded with quercetin prepared in Example 5 (quercetin nanoparticles + calcium 5 mM), the zein / composite polysaccharide nanoparticles loaded with quercetin prepared in Comparative Example 1 (quercetin nanoparticles), and the physical mixture of quercetin / zein / composite polysaccharide prepared in Comparative Example 4 (quercetin physical mixture) were digested with in vitro simulated gastrointestinal digestive juices. The relationship between the percentage of quercetin released from the nanoparticles into the digestive juice and the digestion time during the gastric juice digestion stage and the intestinal juice digestion stage was measured. The percentage of quercetin released into the digestive juice at the end of intestinal digestion was used as the bioavailability of quercetin. The specific method was as follows:

[0139] In vitro simulated gastric digestion stage: The sample was dissolved in 20 mL of ultrapure water to prepare a solution with a quercetin concentration of 0.5 mg / mL. After stirring for 30 min (500 rpm), it was mixed with 20 mL of simulated gastric juice (2 g of sodium chloride and 7 mL of hydrochloric acid were dissolved in 1 L of distilled water, and the pH was adjusted to 1.2), and stirring was continued for 15 min. Then it was placed in a constant temperature water bath at 37 °C for 10 min. After that, the pH of the solution was adjusted to 2.5, 0.064 g of pepsin was added, and it was stirred at a rate of 50 rpm in a constant temperature oscillator at 37 °C for 2 h. The addition of pepsin indicated the start of the gastric digestion process. Samples were taken at 20, 40, 60, 80, and 120 min during digestion, placed in a water bath at 80 °C for 5 min to inactivate pepsin and terminate digestion. Then it was centrifuged at 3000 r / min for 20 min to collect the supernatant, and the quercetin content was detected by ultraviolet spectrophotometry (wavelength 378 nm).

[0140] In vitro simulated intestinal digestion stage: The pH of 40 mL of gastric digestion fluid sample (digested for 2 h) was adjusted to 7.0, 187.5 mg of bile salt was added, and it was placed in a constant temperature water bath at 37 °C. 0.144 g of pancreatin was added, and it was stirred at a rate of 50 rpm in a constant temperature oscillator at 37 °C for 4 h. During digestion, the pH was maintained at 7.0 using an automatic titrator. The addition of pancreatin indicated the start of the intestinal digestion process. Samples were taken at 30 min, 1 h, 2 h, 3 h, and 4 h during digestion, placed in a water bath at 90 °C for 5 min to inactivate pancreatin and terminate digestion. Then it was centrifuged at 3000 r / min for 20 min to collect the supernatant and detect the quercetin content.

[0141] The quercetin release rate (%) was calculated by the following formula:

[0142]

[0143] The experimental results are as Figure 6 、 Figure 7 shown. It can be seen from Figure 6 、 Figure 7 that the release of quercetin mainly occurs in the intestinal digestion stage, and the release rate of quercetin encapsulated in nanoparticles is significantly higher than that of the mixture of quercetin and the raw materials for preparing the particles. The strengthening of calcium ions further delays the release rate of quercetin in the early stage of intestinal digestion, but does not affect the bioavailability of quercetin in the later stage of intestinal digestion.

Claims

1. A protein / polysaccharide nanosphere with intestinal targeting delivery function, characterized in that, the protein / polysaccharide nanosphere is prepared from zein, composite polysaccharide and calcium salt as raw materials; the composite polysaccharide is composed of gum arabic / carrageenan / propylene glycol alginate, pectin and sodium alginate in a mass ratio of 0.1-5:65-75:20-34.9; The preparation method of the protein / polysaccharide nanosphere with intestinal targeting delivery function includes the following steps: S1. Dissolve zein in an ethanol aqueous solution with a volume fraction of 60-90%, so that the concentration of zein is 1-3% w / v to obtain an ethanol solution of zein; S2. Dissolve the composite polysaccharide in water so that the concentration of the composite polysaccharide is 0.1-0.5% w / v, and adjust the pH to 4.0-7.0 to obtain a polysaccharide solution; S3. Disperse the ethanol solution of zein obtained in step S1 into the polysaccharide solution obtained in step S2. The volume ratio of the ethanol solution of zein to the polysaccharide solution is 1:2-1:10, and disperse it by high-speed stirring for 3-8 minutes to obtain a particle dispersion; S4. Rotate and evaporate ethanol from the particle dispersion obtained in step S3, and add water to compensate for the volume of ethanol evaporated to obtain a dispersion; S5. Add calcium salt to the dispersion obtained in step S4 so that the final concentration of calcium ions is 1-50 mM, and stir for 7-15 minutes; S6. Freeze-dry or spray-dry to obtain.

2. The protein / polysaccharide nanosphere with intestinal targeting delivery function according to claim 1, characterized in that, the composite polysaccharide is composed of propylene glycol alginate, pectin and sodium alginate in a mass ratio of 5:75:20; or the composite polysaccharide is composed of carrageenan, pectin and sodium alginate in a mass ratio of 0.1:75:24.9; or the composite polysaccharide is composed of gum arabic, pectin and sodium alginate in a mass ratio of 5:72.5:22.

5.

3. The protein / polysaccharide nanosphere with intestinal targeting delivery function according to claim 1, characterized in that, the calcium salt is calcium chloride or calcium lactate.

4. The protein / polysaccharide nanosphere with intestinal targeting delivery function according to claim 1, characterized in that, when dispersing by high-speed stirring in step S3, the stirring speed is 800-1200 revolutions per minute.

5. Use of the protein / polysaccharide nanosphere with intestinal targeting delivery function according to any one of claims 1-4 in the preparation of an intestinal targeting carrier loaded with drugs.

6. Use of the protein / polysaccharide nanosphere with intestinal targeting delivery function according to any one of claims 1-4 in the preparation of an intestinal targeting carrier loaded with active ingredients.

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