Preparation and Application of OSA-Modified Starch Nanocrystals Microcapsules

Abstract

The present invention belongs to the technical field of the preparation of pharmaceutical microcapsules, and specifically discloses the preparation and application of OSA-modified starch nanocrystal microcapsules, which include the following steps: (1) Using waxy corn starch as a raw material, adding sulfuric acid for hydrolysis reaction to obtain starch nanocrystals; (2) Preparing a starch nanocrystal dispersion liquid, then dropping an OSA-ethanol solution for esterification reaction at room temperature to obtain OSA-modified starch nanocrystals; (3) Mixing the OSA-SNC aqueous dispersion liquid, chitosan, deionized water, and vegetable oil, and using a high-shear dispersion emulsifier to prepare the mixture into an emulsion. Under magnetic stirring, a cross-linking agent is slowly added to the emulsion, and microcapsules are obtained after the cross-linking reaction. The present invention uses waxy corn starch with good biocompatibility as a raw material, and prepares microcapsules with high mechanical strength through the Pickering emulsion templating method. Through in vitro simulated digestion experiments, it can be verified that the active substances in the microcapsules can be targeted and released in the intestine, which has important significance for solving problems in the field of drug delivery.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of pharmaceutical microcapsules, and particularly relates to the preparation and application of OSA-modified starch nanocrystal microcapsules. Background Art

[0002] Intestinal targeted drug delivery has important application value for the treatment of intestinal diseases such as constipation, enteritis, and colorectal cancer. Among them, realizing the controlled release of drugs in the intestine is the key problem to be solved. The emergence of microcapsule technology points out the direction for solving this problem. Microcapsules are substances that separate the internal space and the external space through a film-forming substance to form a specific geometric structure, and have advantages such as controlled release, providing stability to the core material, and shielding taste and odor. The Pickering emulsion templating method is a further development on the traditional microcapsule technology. It is environmentally friendly, has strong stability, and is not easily affected by factors such as the pH value, salt concentration, temperature, and oil phase composition of the system. Therefore, using natural biological materials to make Pickering emulsions has good research prospects. Pickering emulsions are stabilized by colloidal particles. Therefore, selecting colloidal particles with different functions and properties can regulate the size, structure, strength, surface properties, etc. of microcapsules, and have broad application prospects in fields such as cosmetics, biocatalysis, and microreactors. However, there are few research reports on its application in the field of drug loading at present, mainly due to two reasons: (1) The colloidal particles used in microcapsules have poor biocompatibility; (2) The microcapsule wall is relatively thin, with poor mechanical strength and is prone to rupture. Therefore, using a kind of colloidal particle with good biocompatibility to stabilize the emulsion and preparing microcapsules with high mechanical strength by the Pickering templating method has important significance for solving the problems in the field of drug delivery. Summary of the Invention

[0003] The main object of the present invention is to provide an OSA-modified starch nanocrystal microcapsule to solve the problems existing in the background art. The present invention uses waxy corn starch with good biocompatibility as the raw material, and preparing microcapsules with high mechanical strength by the Pickering emulsion templating method has important significance for solving the problems in the field of drug delivery. By simulating the digestion of OSA-modified starch nanocrystals in the human oral cavity, stomach, and intestine in vitro, and observing the changes in their microscopic morphology at the same time, it provides theoretical and experimental basis for its application in industries such as food and biomedicine, and has good economic and social value.

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] A preparation method of an OSA-modified starch nanocrystal microcapsule, comprising the following steps:

[0006] (1) Preparation of starch nanocrystals

[0007] Using waxy corn starch as raw material, adding sulfuric acid solution for hydrolysis reaction, centrifuging and washing with water multiple times to obtain starch nanocrystals with a size of 40 - 200 nm, and storing them refrigerated for later use;

[0008] (2) Preparation of OSA - modified starch nanocrystals

[0009] Preparing a starch nanocrystal aqueous dispersion with a certain concentration, dropping OSA - ethanol solution, and carrying out an esterification reaction at room temperature for 2 - 6 h. During the reaction process, control the pH to be 7.5 - 9.0. After the reaction, centrifuge and wash with alcohol multiple times to obtain OSA - modified starch nanocrystals (OSA - SNC), and store them refrigerated for later use;

[0010] (3) Preparation of microcapsules

[0011] Mixing the OSA - SNC aqueous dispersion, chitosan, deionized water, and vegetable oil, using a high - shear dispersion emulsifier to prepare the mixture into an emulsion. Under the condition of magnetic stirring, slowly add a cross - linker to the emulsion and react for 12 - 24 h to obtain OSA - modified starch nanocrystal microcapsules.

[0012] Furthermore, in step (1), the mass - to - volume ratio of waxy corn starch to sulfuric acid is 1:10 - 1:25, and the concentration of the sulfuric acid is 2.0 - 4.5 mol / L.

[0013] Furthermore, in step (1), the reaction temperature is 20 - 45 °C, stirring at 100 r / min for 2 - 6 d, and centrifuging and washing the reaction product with water multiple times until the pH value is 6 - 7.

[0014] Furthermore, in step (2), the concentration of the OSA - ethanol solution is 2 - 20%, and the concentration of the starch nanocrystal dispersion is 0.2 - 1.0%.

[0015] Furthermore, in step (2), the steps of centrifuging and washing with alcohol are: centrifuging and washing at least 3 times with 80% ethanol aqueous solution and absolute ethanol respectively.

[0016] Optionally, in steps (1) and (2), the preservation method is: adding 0.02% potassium sorbate, preparing an aqueous dispersion of this substance with a certain concentration, and storing it refrigerated at 4 °C for later use.

[0017] Furthermore, in step (3), the concentration of the OSA - SNC aqueous dispersion is 0.5 - 1.5%, the concentration of the chitosan solution is 0.5 - 1.5%, and the volume ratio of the OSA - SNC aqueous dispersion to chitosan is 1:1.

[0018] Optionally, in step (3), the cross - linker is glutaraldehyde, genipin, sodium tripolyphosphate, sodium hexametaphosphate, or vanillin, etc.; the vegetable oil is olive oil, corn oil, peanut oil, or soybean oil, etc.

[0019] Preferably, the concentration of the crosslinking agent is 5-25%, and the volume ratio of the addition amount of the crosslinking agent to the addition amount of the OSA-SNC aqueous dispersion is 1:1.

[0020] In the present invention, waxy corn starch is used as a raw material, starch nanocrystals are prepared by a sulfuric acid hydrolysis method, Pickering emulsions are prepared from starch nanocrystals (OSA-SNC) modified with OSA, and then microcapsules are prepared by an emulsion templating method and their in vitro simulated digestion experiments are explored. The research results show that: the starch nanocrystals prepared by the sulfuric acid hydrolysis method have a particle size of 40-200 nm and a polygonal lamellar structure; infrared characterization reveals that no other groups are introduced into OSA-SNC except for the octenyl succinic acid group, and the charge amount increases with the increase of the degree of substitution (DS); microcapsules are successfully prepared after crosslinking the emulsion stabilized by OSA-SNC + chitosan with glutaraldehyde; the digestion amount of the emulsion stabilized by OSA-SNC + chitosan is the lowest, and the microscopic morphology changes little, which can achieve the experimental expectation of targeted release of active substances in the intestine in the microcapsules. Description of the Drawings

[0021] Figure 1 (A) SEM images of waxy corn starch and (B) SNC;

[0022] Figure 2 Effect diagram of the influence of the addition amount of OSA on the degree of substitution;

[0023] Figure 3 (a) Infrared spectra of SNC and (b) OSA-SNC (DS = 0.036);

[0024] Figure 4 (A) Intact microcapsules and (B) Microscopic images after microcapsule rupture;

[0025] Figure 5 Glucose standard curve graph;

[0026] Figure 6 Effect diagram of the digestion amount in different starch-based in vitro digestion models;

[0027] Figure 7 Microscopic morphology diagrams of the emulsion during digestion; Microscopic images of (A) oral fluid for 3 min; (B) gastric fluid for 60 min; (C) small intestinal fluid for 20 min; (D) small intestinal fluid for 1 h; (E) small intestinal fluid for 2 h; (F) small intestinal fluid for 3 h and (G) small intestinal fluid for 4 h (scale bar is 100 μm). Detailed Embodiments

[0028] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The materials used in the present invention: waxy corn starch, octenyl succinic anhydride, were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; sodium hydroxide, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium sulfate, sodium chloride, sodium bicarbonate, potassium thiocyanate are all of analytical grade; chitosan, glutaraldehyde, urea, uric acid, bile salts, calcium chloride are all of analytical grade; Megazyme GOPOD detection kit was purchased from Hefei Bomei Biotechnology; absolute ethanol, sulfuric acid, hydrochloric acid are all of analytical grade; α-amylase, glucoamylase, pancreatin are all biological reagents, purchased from Tianjin Guangfu Fine Chemical Research Institute.

[0029] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0030] Example 1

[0031] A preparation method of OSA-modified starch nanocrystalline microcapsules, comprising the following steps:

[0032] (1) Preparation of starch nanocrystals (SNC): Using waxy corn starch as the raw material, starch nanocrystals (SNC) were prepared by sulfuric acid hydrolysis. The mass-volume ratio of waxy corn starch to sulfuric acid was 1:10, the concentration of sulfuric acid was 3.16 mol / L, the reaction temperature was 40 °C, and it was stirred at 100 r / min for 6 d. After the reaction ended, the reaction product was centrifuged and washed with water multiple times until the pH value was 6-7, and 0.02% potassium sorbate was added and stored refrigerated for later use.

[0033] (2) Preparation of OSA-modified starch nanocrystals: Prepare a 5% OSA-ethanol solution for later use, then prepare a 0.5% starch nanocrystal (SNC) dispersion. Use a 0.1000 mol / L sodium hydroxide aqueous solution to maintain the system pH value at 8.0. Dropwise add a certain amount of 5% OSA-ethanol solution within 1 h, and always use the sodium hydroxide aqueous solution to maintain the system pH value at 8.0. Carry out the esterification reaction at room temperature for 3 h. After the reaction ended, adjust the system pH value to about 7.0 with hydrochloric acid aqueous solution, and centrifuge and wash 3 times with 80% ethanol aqueous solution and absolute ethanol respectively to obtain OSA-modified starch nanocrystals (OSA-SNC), and the degree of substitution (DS) is 0.019.

[0034] (3) Preparation of microcapsules: According to the volume ratio, 1 part of 1.0% OSA-SNC aqueous dispersion, 1 part of 1.0% chitosan solution, and 5 parts of deionized water were mixed evenly. The pH was adjusted to about 6.0 with NaOH solution, and then 7 parts of olive oil was added as the oil phase. The mixture was prepared into an emulsion with a high-shear dispersion emulsifier at a rotation speed of 8000 rpm for 2 min. Under the condition of magnetic stirring, 25% glutaraldehyde was slowly added to the emulsion. The volume ratio of the addition amount of glutaraldehyde to the addition amount of OSA-SNC aqueous dispersion was 1:1, and the cross-linking reaction was carried out for 12 h to obtain the microcapsules. After reacting for 5 h in the in vitro simulated digestion experiment, the digestion amount of this microcapsule was 3.89%.

[0035] Example 2

[0036] A preparation method of OSA-modified starch nanocrystal microcapsules, comprising the following steps:

[0037] (1) Preparation of starch nanocrystals (SNC): Using waxy corn starch as the raw material, starch nanocrystals (SNC) were prepared by sulfuric acid hydrolysis. The mass-volume ratio of waxy corn starch to sulfuric acid was 1:15, the concentration of sulfuric acid was 3.00 mol / L, the reaction temperature was 30 °C, and it was stirred at 100 r / min for 5 d. After the reaction ended, the reaction product was centrifuged and washed with water multiple times until the pH value was 6 - 7, and 0.02% potassium sorbate was added and stored refrigerated.

[0038] (2) Preparation of OSA-modified starch nanocrystals: Prepare 20% OSA-ethanol solution for later use. Then prepare a 1.0% starch nanocrystal (SNC) dispersion, maintain the pH value of the system at 7.5 with 0.1000 mol / L sodium hydroxide aqueous solution, and gradually add a certain amount of 20% OSA-ethanol solution dropwise within 1 h. Always use the sodium hydroxide aqueous solution to maintain the pH value of the system at 7.5, and carry out the esterification reaction at room temperature for 6 h. After the reaction ended, the pH value of the system was adjusted to about 7.0 with hydrochloric acid aqueous solution, and it was centrifuged and washed 3 times with 80% ethanol aqueous solution and absolute ethanol respectively to obtain OSA-modified starch nanocrystals (OSA-SNC) with a degree of substitution (DS) of 0.015.

[0039] (3) Preparation of microcapsules: According to the volume ratio, 1 part of the 0.5% OSA-SNC aqueous dispersion, 1 part of the 1.5% chitosan solution, and 5 parts of deionized water were mixed evenly. The pH was adjusted to about 6.0 with NaOH solution, and then 7 parts of corn oil was added as the oil phase. The mixture was prepared into an emulsion with a high-shear dispersion emulsifier at a rotation speed of 8000 rpm and an emulsification time of 2 min. Under the condition of magnetic stirring, 5% glutaraldehyde was slowly added to the emulsion. The volume ratio of the addition amount of glutaraldehyde to the addition amount of OSA-SNC aqueous dispersion was 1:1, and the cross-linking reaction was carried out for 24 h to obtain the microcapsules. After reacting for 5 h in the in vitro simulated digestion experiment, the digestion amount of this microcapsule was 5.66%.

[0040] Example 3

[0041] A preparation method of OSA-modified starch nanocrystal microcapsules, comprising the following steps:

[0042] (1) Preparation of starch nanocrystals (SNC): Using waxy corn starch as the raw material, starch nanocrystals (SNC) were prepared by the sulfuric acid hydrolysis method. The mass-volume ratio of waxy corn starch to sulfuric acid was 1:25, the concentration of sulfuric acid was 2.00 mol / L, the reaction temperature was 45 °C, and stirring was carried out at 100 r / min for 4 d. After the reaction ended, the reaction product was centrifuged and washed with water several times until the pH value was 6-7, and 0.02% potassium sorbate was added and stored refrigerated for later use.

[0043] (2) Preparation of OSA-modified starch nanocrystals: Prepare a 2% OSA-ethanol solution for later use, and then prepare a 0.2% starch nanocrystal (SNC) dispersion. Use a 0.1000 mol / L sodium hydroxide aqueous solution to maintain the system pH value at 8.0, and gradually add a certain amount of 2% OSA-ethanol solution dropwise within 1 h. Always use the sodium hydroxide aqueous solution to maintain the system pH value at 9.0, and carry out the esterification reaction at room temperature for 3 h. After the reaction ended, the system pH value was adjusted to about 7.0 with hydrochloric acid aqueous solution, and centrifuged and washed 3 times with 80% ethanol aqueous solution and absolute ethanol respectively to obtain OSA-modified starch nanocrystals (OSA-SNC) with a degree of substitution (DS) of 0.016.

[0044] (3) Preparation of microcapsules: According to the volume ratio, 1 part of the 1.5% OSA-SNC aqueous dispersion, 1 part of the 0.5% chitosan solution, and 5 parts of deionized water were mixed evenly. The pH was adjusted to about 6.0 with NaOH solution, and then 7 parts of olive oil was added as the oil phase. The mixture was prepared into an emulsion with a high-shear dispersion emulsifier at a rotation speed of 8000 rpm for 2 min. Under the condition of magnetic stirring, 10% glutaraldehyde was slowly added to the emulsion. The volume ratio of the added amount of glutaraldehyde to the added amount of OSA-SNC aqueous dispersion was 1:1, and the cross-linking reaction was carried out for 16 h to obtain the microcapsules. After reacting for 5 h in the in vitro simulated digestion experiment, the digestion amount of this microcapsule was 5.12%.

[0045] Example 4

[0046] A preparation method of OSA-modified starch nanocrystal microcapsules, comprising the following steps:

[0047] (1) Preparation of starch nanocrystals (SNC): Using waxy corn starch as the raw material, starch nanocrystals (SNC) were prepared by sulfuric acid hydrolysis. The mass-volume ratio of waxy corn starch to sulfuric acid was 1:20, the concentration of sulfuric acid was 4.50 mol / L, the reaction temperature was 20 °C, and it was stirred at 100 r / min for 2 d. After the reaction, the reaction product was centrifuged and washed with water several times until the pH value was 6 - 7, and 0.02% potassium sorbate was added and stored refrigerated.

[0048] (2) Preparation of OSA-modified starch nanocrystals: Prepare a 15% OSA-ethanol solution for standby, then prepare a 0.8% starch nanocrystal (SNC) dispersion. The pH value of the system was maintained at 8.5 with 0.1000 mol / L sodium hydroxide aqueous solution. A certain amount of 15% OSA-ethanol solution was added dropwise within 1 h, and the pH value of the system was always maintained at 8.0 with sodium hydroxide aqueous solution. The esterification reaction was carried out at room temperature for 3 h. After the reaction, the pH value of the system was adjusted to about 7.0 with hydrochloric acid aqueous solution, and it was centrifuged and washed 3 times with 80% ethanol aqueous solution and anhydrous ethanol respectively to obtain OSA-modified starch nanocrystals (OSA-SNC) with a degree of substitution (DS) of 0.018.

[0049] (3) Preparation of microcapsules: According to the volume ratio, 1 part of the OSA-SNC aqueous dispersion with a mass fraction of 0.5%, 1 part of the chitosan solution with a mass fraction of 1.0%, and 5 parts of deionized water were mixed evenly. The pH was adjusted to about 6.0 with NaOH solution, and then 7 parts of olive oil was added as the oil phase. The mixture was prepared into an emulsion with a high-shear dispersion emulsifier, the rotation speed of the emulsifier was 8000 rpm, and the emulsification time was 2 min. Under the condition of magnetic stirring, 20% glutaraldehyde was slowly added to the emulsion. The volume ratio of the added amount of glutaraldehyde to the added amount of the OSA-SNC aqueous dispersion was 1:1, and the cross-linking reaction was carried out for 18 h to obtain the microcapsules. After reacting for 5 h in the in vitro simulated digestion experiment, the digestion amount of this microcapsule was only 4.34%.

[0050] Experimental Example 1

[0051] 1. Preparation of starch nanocrystals (SNC)

[0052] Using waxy corn starch as the raw material, starch nanocrystals (SNC) were prepared by the sulfuric acid hydrolysis method. Accurately weigh 10 g of waxy corn starch and place it in a 250 mL three-necked flask. Add 100 mL of 3.16 mol / L H2SO4 solution, the reaction temperature is 40 °C, and stir at 100 r / min for 6 d. After the reaction is completed, the reaction product is centrifuged and washed with water multiple times until the pH value is 6-7. A certain concentration of SNC aqueous dispersion (containing 0.02% potassium sorbate) is prepared and stored at 4 °C for later use.

[0053] SNC was prepared by degrading waxy corn starch by the sulfuric acid hydrolysis method. The results of scanning electron microscopy (SEM) measurement are as Figure 1 shown. It can be seen that the shape of waxy corn starch is a rough polygonal structure, and the particle size is in the range of 6-15 μm. The particle size of SNC is much smaller than that of waxy corn starch. The prepared SNC has a polygonal lamellar structure, and the particle size is in the range of 40-100 nm. Not only individual dispersed SNC can be observed, but also some SNC aggregates can be observed to form agglomerated particles with a particle size greater than 100 nm.

[0054] 2. Preparation of OSA-modified starch nanocrystals

[0055] Use a pipette to measure 1.25 mL of octenyl succinic anhydride (OSA) and place it in a 25 mL volumetric flask. Dilute it to the mark with absolute ethanol to obtain an OSA-ethanol solution with a concentration of 5%, and set it aside. Accurately prepare an SNC aqueous dispersion with a concentration of 0.5%. Use a 0.1000 mol / L sodium hydroxide aqueous solution to maintain the pH value of the system at 8.0. Dropwise add a certain amount of the 5% OSA-ethanol solution within 1 h, and always use a 0.1 mol / L sodium hydroxide aqueous solution to maintain the pH value of the system at 8.0. Carry out the esterification reaction at room temperature for 3 h. After the reaction is completed, adjust the pH value of the system to approximately 7.0 with a 0.1 mol / L hydrochloric acid aqueous solution, and centrifuge and wash it 3 times with 80% ethanol aqueous solution and absolute ethanol respectively to obtain octenyl succinic anhydride modified starch nanocrystals (OSA-SNC). Prepare an aqueous dispersion of OSA-SNC with a certain concentration (containing 0.02% potassium sorbate), and place it in a refrigerator at 4 °C for later use.

[0056] Under the conditions of SNC concentration of 0.5%, temperature of 25 °C, system pH value of 8.0, and reaction time of 4 h in this invention, the effect of OSA addition amount on the degree of substitution (DS) was investigated, and the results are shown in Figure 2 . It can be seen that the DS increases linearly with the increase of the OSA addition amount. This is because the higher the OSA addition amount, the greater the contact probability between OSA and SNC, and the DS increases rapidly. However, with the increase of the OSA addition amount, in order to maintain the constant pH value of the reaction system, a large amount of sodium hydroxide aqueous solution needs to be added, which may also lead to the destruction of the SNC structure. Therefore, the addition amount of OSA should not be too high.

[0057] In order to determine whether the octenyl succinic acid group is grafted onto the surface of SNC, in this experiment, SNC and OSA-SNC before and after esterification modification were measured by a Fourier transform infrared spectrometer, as shown in Figure 3 . It can be seen that the peak shapes of the infrared spectra of SNC and OSA-SNC are similar, and there are strong absorptions at 3378, 2932, and 1640 cm -1 . However, compared with the infrared spectrum of SNC, there are two new characteristic absorption peaks in the infrared spectrum of OSA-SNC. The absorption peak at 1726 cm -1 proves the existence of ester carbonyl in the product, indicating that an esterification reaction has occurred between OSA and SNC, which is consistent with the results of OSA modification of native starch. The new characteristic peak appearing at 1574 cm -1 is caused by the asymmetric stretching vibration of RCOO-. The reason is that the esterification reaction is carried out under weak alkaline conditions, and the octenyl succinic acid starch nanocrystal ester exists in the form of sodium salt.

[0058] 3. Preparation of microcapsules

[0059] Accurately pipette 1.00 mL of the OSA-SNC aqueous dispersion with a mass fraction of 1.0% into a 25 mL capped sample bottle, add 1.00 mL of the chitosan solution with a concentration of 1.0% and 5.00 mL of deionized water, mix well, adjust the pH to about 6.0 with 0.1 mol / L NaOH solution, then add 7.00 mL of olive oil as the oil phase, and use a high-shear dispersion emulsifier to prepare the mixture into an emulsion. The rotation speed of the emulsifier is 8000 rpm, and the emulsification time is 2 min. Under the condition of magnetic stirring, slowly add 200 μL of glutaraldehyde with a concentration of 25% to the emulsion, and carry out cross-linking reaction for 2 h to obtain microcapsules.

[0060] The microcapsules prepared from OSA-SNC and chitosan were observed by a super-depth-of-field microscope, as Figure 4 shown. It can be seen that the particle size of the microcapsules is complete and regular, with a diameter in the range of 6 - 40 μm. After waiting for a period of time for the water to evaporate, the microcapsules rupture, leaving a "membrane", indicating that the microcapsules have been successfully prepared.

[0061] Scanning electron microscopy analysis method

[0062] The morphological structures of waxy corn starch and SNC were observed by scanning electron microscopy. An appropriate amount of the sample was evenly spread on the black conductive adhesive on the sample stage. After gold spraying treatment, the sample was photographed and observed by a scanning electron microscope, and the acceleration voltage was 1.0 kV.

[0063] Determination method of degree of substitution

[0064] Accurately measure a certain volume of the OSA-SNC aqueous dispersion and place it in a 50 mL conical flask, add 10 mL of 0.0500 mol / L sodium hydroxide solution, and stir and react for 24 h. Then add 1 - 2 drops of 0.5% phenolphthalein indicator, and titrate with 0.0200 mol / L hydrochloric acid solution until the pink color just fades. Using unmodified SNC as a blank control, read and record the data, and the degree of substitution (DS) is calculated according to formulas (1) and (2).

[0065]

[0066] In the formula: 162 is the molar mass of glucose residue, g / mol;

[0067] 210 is the molar mass of octenyl succinic anhydride, g / mol;

[0068] A is the amount of substance of octenyl succinic anhydride ester group in each gram of OSA-SNC.

[0069] The value of A can be calculated by the following formula:

[0070]

[0071] Where: V is the volume of hydrochloric acid solution required for titrating OSA-SNC, mL;

[0072] V0 is the volume of hydrochloric acid solution required for titrating unmodified SNC, mL;

[0073] M is the molar concentration of hydrochloric acid solution, mol / L;

[0074] W is the mass of the sample, g.

[0075] Determination method of infrared spectrum

[0076] Turn on the dehumidifier to dehumidify for 30 min, install the ATR accessory, turn on the power of the computer and the host of the infrared spectrometer, and preheat for half an hour. Set the test parameters, scan the air background, then closely attach the surface to be measured of the sample to the infrared transparent crystal surface of the ATR accessory, rotate the pressure axis, set the pressure to the standard pressure value required for the experiment, and scan to collect the attenuated total reflection infrared spectrum of the sample. After the test is completed, wipe the ATR indenter and other accessories with absolute ethanol, and keep the sample chamber clean.

[0077] Experimental Example 2

[0078] 1. Construction of in vitro simulated digestion model

[0079] According to the structure of the human digestive tract, the composition of digestive juices, and the digestion characteristics of oils and starches, an in vitro digestion model is constructed. The in vitro digestion model includes three stages: simulated saliva fluid (SSF), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF).

[0080] 1.1 Simulated oral stage

[0081] Prepare fresh SSF: Weigh 0.0358 g of KCl, 0.0355 g of NaH2PO4, 0.0288 g of Na2SO4, 0.0119 g of NaCl, 0.0678 g of NaHCO3, 0.008 g of urea, 0.0006 g of uric acid, and 0.024 g of α-amylase in a 100 mL beaker, add 40 mL of deionized water, and stir to dissolve. Pipette 4.00 mL of SSF into a conical flask, then add 4.00 mL of the mixed OSA-SNC microcapsule emulsion, adjust the pH to about 7, set the temperature to 37 °C, the rotation speed to 100 rpm, and react in a water bath thermostatic shaker for 3 min.

[0082] 1.2 Simulated gastric stage

[0083] Prepare fresh SGF: Weigh 0.1000 g of NaCl into a 100 mL beaker, add 350 μL of concentrated hydrochloric acid, transfer it to a 50 mL volumetric flask, make up the volume to the mark and shake well. After oral digestion is completed, pipette 10.00 mL of SGF into the digestion system, adjust the pH value of the system to about 2 with 0.5000 mol / L NaOH solution, and react in a water bath thermostatic shaker for 1 h. Set the temperature at 37 °C and the rotation speed at 100 rpm.

[0084] 1.3 Simulated small intestine stage

[0085] Prepare 15 mL of fresh SIF: It includes 1 mL of CaCl2 (165 mM), 13.2 mL of a mixed solution of pancreatin (2.5 - 9 mg / mL) and bile salts (50 mg / mL), 20 μL of glucoamylase (0.1188 mg / mL) and 780 μL of deionized water.

[0086] After the gastric stage is completed, adjust the pH value of the system to about 7 with 1.0 mol / L NaOH solution. Add 15.00 mL of simulated small intestine fluid to the digestion system, set the temperature at 37 °C, and simulate the digestion of small intestine fluid at 100 rpm for 4 h. During this stage, 0.5 mol / L NaOH solution needs to be continuously added to the system to maintain the pH value at about 7.

[0087] 2. In vitro simulated digestion experiment of microcapsules

[0088] 2.1 Drawing of glucose standard curve

[0089] Pipette 0.00, 0.04, 0.08, 0.12, 0.16 and 0.20 mL of 1.0 mg / mL glucose standard working solution into colorimetric tubes respectively, then add 0.20, 0.16, 0.12, 0.08, 0.04 and 0.00 mL of deionized water respectively. Finally, add 6 mL of GOPOD reagent, shake well, incubate at 40 - 50 °C for 20 min, use deionized water as the reference, and measure the absorbance at the maximum absorption wavelength. Draw the glucose standard curve with the absorbance as the ordinate and the corresponding glucose content (mg / mL) as the abscissa.

[0090] 2.2 Determination of starch-based digestion amount

[0091] During the in vitro simulated digestion process, 200 μL of samples were taken at 3 min of simulated oral cavity, 1 h of simulated stomach, 20 min, 60 min, 120 min, 180 min, and 240 min of simulated intestine. After inactivating the enzymes of the samples in boiling water for 5 min, they were centrifuged, and the supernatant was taken to determine the glucose content by the glucose oxidase / peroxidase (GOPOD) method. That is, 200 μL of the supernatant was added to a 10 mL colorimetric tube, and then 6 mL of GOPOD reagent was added. After shaking well, it was incubated at 40 - 50 °C for 20 min. Using deionized water as the reference, the absorbance was measured at the maximum absorption wavelength. The digestion amount of OSA-SNC was calculated according to formula (3).

[0092]

[0093] Where: M i is the glucose content in the in vitro simulated digestion system, g;

[0094] M 总 is the total mass of OSA-SNC in the system, g;

[0095] 0.9 is the stoichiometric constant for the conversion of glucose to starch.

[0096] 2.3 Determination of the microscopic morphology of the emulsion during digestion

[0097] During the in vitro simulated digestion process, 500 μL of samples were taken at 3 min of simulated oral cavity, 1 h of simulated stomach, 20 min, 60 min, 120 min, 180 min, and 240 min of simulated intestine, and the microscopic morphology of the emulsion was observed with a microscope. Specific process: Place 1 drop of water on a glass slide, add an appropriate amount of emulsion and disperse it evenly. Using a bottom light source, place it in the optical path of a super-depth-of-field microscope for observation. The magnification can be adjusted as needed, photographed and saved, and the change in the emulsion particle size was analyzed according to the following formula.

[0098]

[0099]

[0100]

[0101]

[0102] Where: Di is the diameter of a single milk droplet, μm; Dn is the average diameter of the droplets, μm; D 3,2 is the surface area average diameter of the droplets, μm; D 4,3 is the volume average diameter of the droplets, μm; N is the number of droplets used in the calculation (N≥50); PDI is the dispersion index.

[0103] 3. Results and Discussion

[0104] 3.1 Plotting of the Standard Curve

[0105] The standard curve of glucose was plotted according to the method in 2.1, and the results are as Figure 5 shown. By performing linear fitting on it, the regression equation of the standard curve can be obtained as: y = 33.191x - 0.0005, R 2 = 0.9992; this working curve has a good linear relationship in the range of 0 - 0.032 mg / mL, and the content of glucose can be calculated.

[0106] 3.2 Result Analysis of the Digestibility of Starch-based Substances

[0107] The digestibility of starch-based substances in waxy corn starch, rice starch, SNC + chitosan microcapsules, and OSA - SNC + chitosan microcapsules in the in vitro simulated digestion experiment was measured, and the results are as Figure 6 shown. It can be seen that the digestibility of the four starch-based substances in the simulated oral cavity and gastric juice is almost zero, probably because the concentration of α-amylase in the oral cavity is low and the digestion time is short, and there is no amylase in the gastric juice to hydrolyze the glycosidic bonds of the starch-based substances. After entering the simulated small intestine stage, the digestibility of the four starch-based substances increases, but the increase amounts are different. The order of digestibility from large to small is rice starch, waxy corn starch, SNC + chitosan microcapsules, and OSA - SNC + chitosan microcapsules. Among them, the digestibility of OSA - SNC + chitosan microcapsules is the lowest. After reacting for 5 h in the in vitro simulated digestion experiment, the digestibility is only 3.89%, indicating that the digestibility of starch can be effectively reduced by OSA modification. The results show that: OSA-modified starch nanocrystal microcapsules can maintain the state of microcapsules in the in vitro simulated digestion experiment, achieve the purpose of targeted release, and meet the experimental expectations.

[0108] 3.3 Microscopic Morphology of the Emulsion during Digestion

[0109] The microscopic morphology of the emulsion during digestion was observed according to the method in 2.3, and the results are as Figure 7 and Table 1 shown. It can be seen that with the increase of the reaction time, the particle size of the microcapsules in the emulsion changes little. After simulating small intestine digestion for 240 min, the average particle size D n of the emulsion only increases from 20.15 μm in the oral cavity to 26.80 μm, indicating that the microcapsules have good stability during the in vitro simulated digestion experiment, laying a good foundation for the release of active substances in the oil phase at the intestinal site.

[0110] Table 1 Results of the Particle Size Change of the Emulsion in the In Vitro Simulated Digestion Experiment

[0111]

[0112]

[0113] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of OSA-modified starch nanocrystal microcapsules, characterized in that, It includes the following steps: (1) Preparation of starch nanocrystals Using waxy corn starch as raw material, adding sulfuric acid solution for hydrolysis reaction, centrifuging and washing with water for multiple times to obtain starch nanocrystals with a size of 40 - 200 nm, and storing them in refrigeration for later use; (2) Preparation of octenyl succinic anhydride (OSA) - modified starch nanocrystals Preparing a starch nanocrystal aqueous dispersion with a certain concentration, dropping OSA - ethanol solution for esterification reaction at room temperature for 2 - 6 h, controlling the pH at 7.5 - 9.0 during the reaction, centrifuging and washing with alcohol for multiple times after the reaction to obtain OSA - modified starch nanocrystals (OSA - SNC), and storing them in refrigeration for later use; (3) Preparation of microcapsules Mixing the OSA - SNC aqueous dispersion, chitosan solution, deionized water and vegetable oil evenly, using a high - shear dispersion emulsifier to prepare the mixture into an emulsion, and slowly adding a cross - linker to the emulsion under magnetic stirring for reaction for 12 - 24 h to obtain OSA - modified starch nanocrystal microcapsules; In step (3), the concentration of the OSA - SNC aqueous dispersion is 0.5 - 1.5%, the concentration of the chitosan solution is 0.5 - 1.5%, and the volume ratio of the OSA - SNC aqueous dispersion to the chitosan solution is 1:1; The cross - linker is glutaraldehyde; the concentration of the cross - linker is 5 - 25%, and the volume ratio of the addition amount of the cross - linker to the addition amount of the OSA - SNC aqueous dispersion is 1:

1.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass - to - volume ratio of waxy corn starch to sulfuric acid is 1:10 - 1:25, and the concentration of the sulfuric acid is 2.0 - 4.5 mol / L.

3. The preparation method according to claim 1, characterized in that, In step (1), the reaction temperature is 20 - 45 °C, stirring at 100 r / min for 2 - 6 d, and centrifuging and washing the reaction product with water for multiple times until the pH value is 6 - 7.

4. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the OSA - ethanol solution is 2 - 20%, and the concentration of the starch nanocrystal dispersion is 0.2 - 1.0%.

5. The preparation method according to claim 1, characterized in that, In step (2), the steps of centrifuging and washing with alcohol are: centrifuging and washing at least 3 times with 80% ethanol aqueous solution and absolute ethanol respectively.

6. The preparation method according to any one of claims 1-5, characterized in that, In steps (1) and (2), adding 0.02% potassium sorbate, preparing an aqueous dispersion of this substance with a certain concentration, and storing it in refrigeration at 4 °C for later use.

7. The preparation method according to any one of claims 1-5, characterized in that, In step (3), the vegetable oil is olive oil, corn oil, peanut oil or soybean oil.

8. Application of the microcapsules prepared by the preparation method according to claim 7 in the preparation of intestinal-targeted drugs.

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