Calcium alginate microgels for simultaneous delivery of amphiphilic active substances, preparation method and application thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and discloses a calcium alginate microgel for simultaneously delivering amphiphilic active substances, a preparation method thereof and applications. In the present invention, hydrophobic RES is encapsulated into SNP to form R-SNPs, so as to improve the solubility and stability of RES. Further, R-SNPs and hydrophilic OPC are co-encapsulated into the calcium alginate microgel to achieve the targeted delivery of the two active substances, which not only has good drug release performance but also can avoid the possible nano-toxicity of nanoparticles. The constructed microgel provides a new direction for the targeted delivery of active substances and opens up new prospects for alleviating ulcerative colitis.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a calcium alginate microgel for simultaneously delivering amphiphilic active substances, a preparation method thereof and an application thereof. Background Art

[0002] Inflammatory bowel disease is an idiopathic disease caused by a dysregulated immune response of the body to the host gut microbiota, leading to chronic recurrent intestinal inflammation. Ulcerative colitis (UC) is one of the two main types of inflammatory bowel disease. UC causes inflammation and ulcers on the surface of the intestinal mucosa. The mucositis starts from the rectum and extends to the proximal colon in a continuous state. The most common symptoms include chronic recurrent diarrhea, bloody stools, abdominal pain and inflammatory nature, and it can be diagnosed by colonoscopy and histological examination. The histopathological features of UC include severe infiltration of neutrophils and lymphoplasmacyte in the mucosa, destruction and deformation of epithelial crypts, cryptitis and crypt abscesses, and extensive mucosal erosion. UC is common in North America and Europe and has the highest incidence in developed and urbanized areas. Epidemiological surveys show that the number of patients in Europe and the United States accounts for 0.5% of the world's population. In recent years, the incidence and prevalence of UC in Western developed countries have begun to stabilize. However, in developing countries, especially in South America and East Asia, its incidence and prevalence have increased. For example, in the early 1990s, the incidence in Japan had reached 1.95 cases per 100,000 people. Due to the high incidence of UC in developed countries and the significant increase in its incidence in developing countries, it has become a global public health burden.

[0003] Resveratrol (RES) is a polyphenolic stilbene with a double bond connecting two phenolic rings. The biological activities of RES have been widely recognized, such as anti-cancer, inflammation regulation, heart protection, blood sugar lowering, antioxidant, antiviral and antifungal, and phytoalexin activities, etc. In biological systems, RES is rapidly and widely metabolized. In order to exert the beneficial effects of RES, its concentration in the blood must reach at least 10 mg / L. For an average-weight person, this is equivalent to needing to absorb 50 mg of RES to exert its efficacy. However, the solubility of RES in water is very low, about 0.03 mg / mL, and it is difficult to reach its effective dose by directly ingesting foods containing RES. In addition, the gastrointestinal stability of RES is poor, seriously affecting its absorption and bioavailability. At the same time, RES has been proven to be effective for UC, but its oral bioavailability is low due to its rapid absorption and extensive metabolism in the upper gastrointestinal tract. Therefore, when using RES to relieve UC, a colon-targeted drug delivery system for RES needs to be constructed to overcome the disadvantages of its low solubility, limited stability, and high metabolic rate in the upper gastrointestinal tract, so as to improve the curative effect and reduce adverse reactions.

[0004] Oligomeric proanthocyanidins (OPC) are oligomers composed of flavanol monomers and are widely present in plants such as fruits, seeds, flowers, nuts, and tree barks. OPC has been widely studied for its health-protective activities. These activities include antibacterial activity against pathogens, inhibition of digestive enzymes and enzymes involved in inflammatory responses, general antioxidant activity, improvement of diabetes, reduction of inflammation, improvement of cardiovascular function, and inhibition of carcinogenesis. The bioavailability of OPC is related to its molecular size. The bioavailability of flavan-3-ol monomers is 5-50%, while the absorption of oligomeric proanthocyanidins is slower and lower than that of monomeric flavan-3-ols. OPC has low bioavailability and limited degradation in the small intestine, and a large part of it is metabolized by the colonic microbiota, that is, most OPCs seem to be excreted as microbial-derived metabolites or phase II conjugates of microbial metabolites. A small part of the unmetabolized OPC dimers is excreted through urine. OPC has been shown to relieve colitis symptoms in animal models. In addition, studies have found that adding OPC to the diet of colitis mice can improve inflammatory bowel disease indicators, change the expression of tight junction proteins, increase the number of colonic goblet cells, and reduce the MPO level. It is also possible to relieve the treatment of colitis by improving the inflammatory response, inhibiting inflammatory cell infiltration and antioxidant damage, promoting the repair of damaged tissues, improving colonic oxidative stress, inhibiting the activity of inducible nitric oxide synthase, and reducing the production of nitric oxide.

[0005] Shellac is a natural resin with good biocompatibility and edibility and is approved by the US Food and Drug Administration (FDA) as a generally recognized safe additive. In addition, it has pH responsiveness, being insoluble under acidic conditions and soluble under slightly alkaline (pH > 7.0) conditions; therefore, it can respond to the different pH environments of the human digestive tract. Shellac nanoparticles (SNP) have a high specific surface area and high loading efficiency, and can load more active substances such as RES. However, SNP is unstable under acidic conditions and is prone to obvious aggregation, which makes SNP not suitable for oral delivery of active substances. Therefore, a multi-level structure delivery system is of great significance for achieving colon-controlled release and multi-drug combination. Summary of the Invention

[0006] To overcome the above-mentioned drawbacks and deficiencies of the prior art, the primary object of the present invention is to provide a method for preparing calcium alginate microgels for simultaneously delivering amphiphilic active substances. This method innovatively encapsulates hydrophobic RES into SNP to form R-SNPs, improving the solubility and stability of RES. Further, by co-encapsulating R-SNPs and hydrophilic OPC into calcium alginate microgels, targeted delivery of two active substances with opposite polarities is achieved. This not only exhibits good drug release properties but also circumvents the potential nanotoxicity of nanoparticles. The constructed microgels provide a new direction for the targeted delivery of active substances with opposite polarities and open up new prospects for alleviating ulcerative colitis.

[0007] Another object of the present invention is to provide calcium alginate microgels prepared by the above method for simultaneously delivering amphiphilic active substances, which have a significant alleviating effect on ulcerative colitis.

[0008] A further object of the present invention is to provide the application of the above calcium alginate microgels for simultaneously delivering amphiphilic active substances in the preparation of drugs for alleviating ulcerative colitis.

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

[0010] A method for preparing calcium alginate microgels for simultaneously delivering amphiphilic active substances, comprising the following steps:

[0011] (1) Dissolve shellac and resveratrol in an anhydrous ethanol solution;

[0012] (2) Filter to remove insoluble substances in the solution of step (1);

[0013] (3) Drop the filtrate obtained in step (2) into water to obtain a dispersion of shellac alcohol nanoparticles loaded with resveratrol;

[0014] (4) Filter and evaporate the dispersion obtained in step (3) to obtain a concentrated dispersion with ethanol removed;

[0015] (5) Add water to the concentrated dispersion in step (4) to obtain a dispersion of shellac nanoparticles;

[0016] (6) Take the dispersion obtained in step (5) and dissolve procyanidins in it;

[0017] (7) Add sodium alginate to the solution obtained in step (6) under stirring to obtain a sol;

[0018] (8) Centrifuge to remove air bubbles in the sol obtained in step (7);

[0019] (9) Drop the sol obtained in step (8) into a CaCl2 solution through a microfluidic device to form microgels;

[0020] (10) Let the microgel stand for curing to obtain calcium alginate microgel.

[0021] In step (1), the mass ratio of shellac to resveratrol is 9:1 to 4:1.

[0022] In step (1), the amount of absolute ethanol used is such that for every 60 mg of resveratrol, 20 - 45 mL of absolute ethanol solution is added.

[0023] In step (2), the diameter of the microporous filter membrane used for filtration is 0.22 - 0.45 μm.

[0024] In step (3), the dropping rate is 0.3 - 0.6 mL / min, and the inner diameter of the needle used is 0.2 - 0.3 mm.

[0025] In step (4), the evaporation in the filtration evaporation is preferably carried out using a rotary evaporator (R - 1001VN, Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd.), and the evaporation parameters are 80 - 120 MPa, 50 - 70 r / min, 45 - 55 °C; the filter paper for filtration is slow filter paper.

[0026] In step (5), the amount of water added satisfies that the concentration of the obtained shellac nanoparticle dispersion is 3 - 6 g / L.

[0027] In steps (6) and (7), the mass ratio of procyanidin (OPC), shellac alcohol nanoparticles loaded with resveratrol (R - SNPs), and sodium alginate (SA), that is, the ratio of OPC:R - SNPs:SA is 1:3:7 to 1:10:40.

[0028] In step (7), the stirring refers to stirring with a mechanical stirrer, and the stirring speed is 200 - 400 r / min.

[0029] In step (8), the centrifugation refers to centrifuging in a high - speed centrifuge (3 - 30k, Germany Sigma) at 1000 - 1500 r / min for 10 - 20 min.

[0030] In step (9), dropping the sol into the CaCl₂ solution through a microfluidic device is specifically operated as follows: using an injection pump (Baoding Shenchen Pump Industry Co., Ltd.) to drop the prepared sol into a 1.5 - 2.5% (m / v, g / mL) CaCl₂ solution with a needle having an inner diameter of 0.25 - 0.35 mm at a speed of 2 - 5 mL / h. Among them, the mass ratio of SA in the sol to the mass of CaCl₂, that is, the ratio of SA:CaCl₂ is 1:5 to 1.5:1.

[0031] In step (10), the standing and curing time is 20 - 40 min.

[0032] A calcium alginate microgel for simultaneously delivering amphiphilic active substances prepared by the above method.

[0033] The calcium alginate microgel for simultaneously delivering amphiphilic active substances achieves the purpose of alleviating ulcerative colitis in terms of improving weight loss, rising DAI, colon atrophy, elevated spleen index, crypt loss, goblet cell loss and neutrophil infiltration in the colon tissue.

[0034] The mechanism of the present invention is as follows:

[0035] First, the present invention loads RES onto SNP by the anti-solvent precipitation method to improve the solubility and stability of RES, and then by the droplet method, that is, under calcium ion crosslinking, SNP loaded with RES (R-SNPs) and OPC are co-loaded into the calcium alginate microgel. The obtained microgel has a multi-level structure of nanoscale and micron-scale. After entering the stomach (acidic condition), the Ca of the calcium alginate microgel 2+ will be replaced by H + to form insoluble alginic acid, which will further aggregate to protect R-SNPs and OPC from being destroyed. Although the microgel will rapidly swell in the small intestine environment with pH = 6.8, it can still maintain an intact structure. When the microgel enters the weakly alkaline (pH = 7.4) colon environment, it will be converted into soluble alginate, and there is no longer a crosslinked structure between the alginic acid molecular chains, which is manifested as the microgel rupturing and R-SNPs and OPC being released. R-SNPs can dissolve shellac under the action of microorganisms and pH (>7.0) to release RES.

[0036] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0037] (1) By encapsulating resveratrol with shellac, shellac nanoparticles are formed. The encapsulation efficiency of R-SNPs in the present invention is 99.03%, indicating that the effective encapsulation of hydrophobic active substances is achieved to the greatest extent.

[0038] (2) By crosslinking shellac nanoparticles and procyanidins with sodium alginate, microgels are formed, which can be applied to the field of medical technology. The drug release rate of the microgels of the present invention in gastric juice is less than 15%, indicating that the microgels can effectively resist the digestion of gastric juice and reach the intestine smoothly.

[0039] (3) The encapsulation of resveratrol and procyanidins realizes the synergistic effect of hydrophilic and hydrophobic active substances, opening up a new way for the use of active substances with opposite polarities.

[0040] (4) In the current research on calcium alginate microgels, there is no report on simultaneously delivering resveratrol and procyanidins. Description of the Drawings

[0041] Figure 1 SEM image (left) and TEM image (right) of R-SNPs in Example 1;

[0042] Figure 2 SEM results of the surface of the gel embedding R-SNPs and the gel without embedding R-SNPs in Example 1 at 100x, 1000x, and 10000x magnifications;

[0043] Figure 3 Swelling ratios (left) of SNPs-GEL, R-SNPs-GEL, SNPs-P-GEL, and R-SNPs-P-GEL after swelling in SGF and SIF for 48 h, and states (right) after swelling in SCF for 48 h;

[0044] Figure 4 Process diagram of the swelling to dissolution of R-SNPs-P-GEL in Example 2;

[0045] Figure 5 Cumulative release curves of RES (left) and OPC (right) in R-SNPs-P-GEL in Example 2;

[0046] Figure 6 Changes in body weight (A) and DAI (B) of mice in each experimental group over time during the entire treatment period in Example 3;

[0047] Figure 7 Colon photos (A) and colon lengths (B) of mice in Example 3;

[0048] Figure 8 Representative images of the colon of mice in each group stained with H&E in Example 3;

[0049] Figure 9 Spleen photos (A) and spleen indices (B) of mice in Example 3. Detailed implementation manners

[0050] The following further describes the present invention in detail in combination with examples and drawings, but the implementation manners of the present invention are not limited thereto. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0051] Reagents used in the examples can be conventionally purchased from the market without special instructions.

[0052] In the present invention, the preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances mainly includes the steps of preparing R-SNPs by using the reverse solvent precipitation method and then preparing R-SNPs-P-GEL by using the droplet method.

[0053] Example 1

[0054] 1. Prepare R-SNPs-P-GEL.

[0055] (1) Dissolve 540 mg of shellac and 60 mg of resveratrol in 20 mL of anhydrous ethanol solution with a concentration of 99.5% (mL / mL);

[0056] (2) Use a 0.45 μm microporous filter membrane to filter out the insoluble substances in the solution of step (1);

[0057] (3) Introduce the filtrate obtained in step (2) into an HL-2B digital display constant current pump, and use a needle with an inner diameter of 0.25 mm to drop it into 100 mL of deionized water at a speed of 0.45 mL / min to obtain a dispersion of shellac alcohol nanoparticles loaded with resveratrol;

[0058] (4) Filter the dispersion obtained in step (3) with slow filter paper, and use a rotary evaporator (R-1001VN, Zhengzhou Great Wall Scientific and Industrial Co., Ltd.) to evaporate and remove ethanol at 100 MPa, 50 °C, and 60 r / min to obtain a concentrated nano-dispersion;

[0059] (5) Add deionized water to the concentrated dispersion in step (4) until the total volume is 100 mL to obtain a dispersion of shellac nanoparticles loaded with resveratrol;

[0060] (6) Take 40 mL of the solution in step (5), and dissolve 80 mg of procyanidins in this solution;

[0061] (7) Add 0.6 g of sodium alginate to the solution obtained in step (6) under mechanical stirring at 300 r / min to obtain a sol;

[0062] (8) Centrifuge at a speed of 1000 r / min for 10 min to remove the bubbles in the sol obtained in step (7);

[0063] (9) Drop the sol obtained in step (8) into 40 mL of 2% (g / mL) CaCl2 solution through a microfluidic device using a needle with an inner diameter of 0.31 mm at a speed of 5 mL / h to form a microgel;

[0064] (10) Let it stand for 30 min to solidify the microgel to obtain calcium alginate microgel.

[0065] 2. After freeze-drying the dispersion of shellac nanoparticles in step (5), observe its surface morphology and microstructure by scanning electron microscopy and transmission electron microscopy, and the results are as Figure 1As shown. It can be seen from the scanning electron microscope image that R-SNPs have a spherical structure and a smooth surface, and the particle sizes are very uniform. It can be found from the transmission electron microscope image that the R-SNPs in the figure have a bilayer structure and an obvious core, indicating the successful loading of RES.

[0066] 3. Freeze-dry the gel embedding R-SNPs and the gel without embedding R-SNPs (i.e., the solution in step (5) is not added in step (6)) in step (10), and observe their surface morphologies under a scanning electron microscope at magnifications of 100 times, 1000 times, and 10000 times. The results are as Figure 2 shown. It can be seen from the figure that the surface of the gel containing R-SNPs is smoother than that of the gel without R-SNPs. Both surfaces have obvious grooves, and the latter has more and deeper grooves. It shows that the addition of R-SNPs may change the gel properties by changing the gel structure, making the network structure of sodium alginate more compact.

[0067] Example 2:

[0068] 1. Prepare R-SNPs-P-GEL.

[0069] (1) Dissolve 520 mg of shellac and 65 mg of resveratrol in 20 mL of an anhydrous ethanol solution with a concentration of 99.5% (mL / mL);

[0070] (2) Filter the solution in step (1) through a 0.22 μm microporous filter membrane to remove insoluble substances;

[0071] (3) Introduce the filtrate obtained in step (2) into an HL-2B digital display constant current pump, and use a needle with an inner diameter of 0.28 mm to drop it into 110 mL of deionized water at a speed of 0.5 mL / min to obtain a dispersion of shellac alcohol nanoparticles loaded with resveratrol;

[0072] (4) Filter the dispersion obtained in step (3) with a slow filter paper, and evaporate and remove ethanol with a rotary evaporator (R-1001VN, Zhengzhou Great Wall Scientific and Industrial Co., Ltd.) at 90 MPa, 48 °C, and 65 r / min to obtain a concentrated nano-dispersion;

[0073] (5) Add deionized water to the concentrated dispersion in step (4) until the total volume is 110 mL to obtain a dispersion of shellac nanoparticles loaded with resveratrol;

[0074] (6) Add 80 mg of OPC to 50 mL of the R-SNPs dispersion and mix well until fully dissolved. While stirring with a mechanical stirrer (350 r / min), slowly add 0.8 g of SA. After it is dissolved into a homogeneous sol, centrifuge it at 1500 r / min in a high-speed centrifuge for 15 min to remove air bubbles. Drop the prepared sol into 40 mL of 2.5% (g / mL) CaCl2 solution at an extrusion rate of 3 mL / h using a needle with an inner diameter of 0.26 mm to form a gel (R-SNPs-P-GEL), and let it stand for 30 min to solidify the microgel.

[0075] (7) Prepare SNPs-GEL in the same way, and use the anti-solvent precipitation method to prepare SNPs. Specifically, dissolve 450 mg of shellac in 20 mL of absolute ethanol with a concentration of 99.5% (mL / mL) at a stirring speed of 300 r / min to obtain a shellac ethanol solution, and then ultrasonicate it for 2 min at 500 W to completely dissolve the shellac. After removing a small amount of insoluble shellac through a 0.45 μm microporous filter membrane, use a needle with an inner diameter of 0.25 mm and a speed of 10 r / min to drop the filtered shellac ethanol solution into 100 mL of deionized water through a constant flow pump. To ensure that almost all of the shellac is converted into SNPs, stir the deionized water at a speed of 350 r / min during the experiment until the shellac ethanol solution is completely dropped. Filter the original SNPs dispersion with slow filter paper to remove a small amount of aggregates, and then evaporate the ethanol from the original dispersion using a rotary evaporator. Add deionized water to the concentrated dispersion to ensure that the final volume of the dispersion is 100 mL. While stirring the 50 mL SNPs dispersion with a mechanical stirrer (350 r / min), slowly add 0.8 g of SA. After it is dissolved into a homogeneous sol, centrifuge it at 1500 r / min in a high-speed centrifuge for 15 min to remove air bubbles. Drop the prepared sol into 40 mL of 2.5% (g / mL) CaCl2 solution at an extrusion rate of 3 mL / h using a needle with an inner diameter of 0.26 mm to form a gel, and let it stand for 30 min to solidify the microgel.

[0076] (8) Similarly, prepare SNPs-P-GEL, that is, add 80 mg of OPC to 50 mL of the SNPs dispersion (prepared in step (7) of Example 2) and mix well until fully dissolved. While stirring with a mechanical stirrer (350 r / min), slowly add 0.8 g of SA. After it is dissolved into a homogeneous sol, centrifuge it at 1500 r / min in a high-speed centrifuge for 15 min to remove air bubbles. Drop the prepared sol into 40 mL of 2.5% CaCl2 solution at an extrusion rate of 3 mL / h using a needle with an inner diameter of 0.26 mm to form a gel, and let it stand for 30 min to solidify the microgel.

[0077] (9) Similarly, prepare R-SNPs-GEL, that is, while stirring 50 mL of R-SNPs dispersion (prepared in step (5) of Example 2) with a mechanical stirrer at 350 r / min, slowly add 0.8 g of SA. After it is dissolved into a uniform sol, centrifuge it at 1500 r / min in a high-speed centrifuge for 15 min to remove air bubbles. Drop the prepared sol into 40 mL of 2.5% CaCl2 solution at an extrusion speed of 3 mL / h with a needle having an inner diameter of 0.26 mm to form a gel, and let it stand for 30 min to solidify the microgel.

[0078] 2. The weight analysis method is used to measure the swelling ratio of calcium alginate microgels that simultaneously deliver amphiphilic active substances, and to investigate the swelling effect of calcium alginate microgels that simultaneously deliver amphiphilic active substances in simulated colon fluid (SCF). The method for measuring the swelling ratio is as follows: Weigh a certain mass of freeze-dried hydrogel precisely in a beaker. Pour 20 mL of simulated gastric fluid (SGF) into the first group of beakers, 20 mL of simulated intestine fluid (SIF) into the second group of beakers, and 20 mL of simulated colon fluid (SCF) into the third group of beakers. According to the Chinese Pharmacopoeia 2005 edition, artificial gastric juice: Take 16.4 mL of dilute hydrochloric acid with a concentration of 1 mol / mL, add 800 mL of water and 10 g of pepsin, shake well, and then dilute with water to 1000 mL. Artificial small intestine fluid: Take 6.8 g of potassium dihydrogen phosphate, add 500 mL of water to dissolve it, adjust the pH value to 6.8 with 0.1 mol / L sodium hydroxide solution. Take 10 g of pancreatin, add an appropriate amount of water to dissolve it. After mixing the two liquids, dilute with water to 1000 mL. Artificial colon fluid: Dissolve 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate in 1000 mL of water. Let the hydrogel swell in the beaker. After swelling for 48 h to equilibrium, then carefully filter out the swollen gel beads from the beaker, dry the surface moisture with filter paper and weigh. The specific formula for calculating the swelling ratio is:

[0079]

[0080] In the formula, W Eq represents the mass (g) of the hydrogel at swelling equilibrium, W d represents the mass (g) of the freeze-dried hydrogel, that is, the mass before swelling, and SW Eq represents the equilibrium swelling ratio of the hydrogel. The results are as Figure 3 shown. The specific swelling process is shown in Figure 4The swelling behavior of polymers is not only determined by the composition of the polymers, but also affected by physical tension and the elastic ability between macromolecular chains. The swelling behaviors of SNPs-GEL, R-SNPs-GEL, SNPs-P-GEL and R-SNPs-P-GEL in SCF and SIF are as Figure 3 shown in the left figure in it. It can be seen that the microspheres do not swell significantly in SGF, while they swell significantly in SIF. Figure 3 The right figure shows the situation of the microspheres in SCF, which is a partial view of the dissolved hydrogel in the beaker. It can be seen that the microspheres have dissolved in the colon fluid. This is because in an acidic environment (SGF), -COO- is converted into -COOH and exists in this form. The intermolecular repulsive force is small, and the microspheres wrap less water, so the swelling rate is low. In a weakly acidic environment (SIF), the internal network structure of the microspheres absorbs water and swells sufficiently. In a weakly alkaline environment (SCF), OH- dissociates -COOH, the intermolecular cross-linking structure is lost, the repulsive force increases, and the molecular chains can diffuse freely in the solution, showing that the microspheres dissolve. Figure 4 shows the process of R-SNPs from swelling to dissolution. By comparing the swelling situations of different microspheres in SIF, it can be known that the swelling rate of the microspheres containing RES or OPC is smaller than that of the microspheres without RES or OPC. This may be because the structure of the alginate microspheres after adding RES or OPC is more compact.

[0081] 3. The drug release behaviors of calcium alginate microgels simultaneously delivering amphiphilic active substances in SGF, SIF and SCF were studied by in vitro experiments. At a constant temperature of 37 °C, 10 mg of R-SNPs-P-GEL was immersed in 10 mL of SGF, SIF and SCF respectively and shaken at a speed of 60 r / min. Next, 200 μL of the above solution was taken out at predetermined time intervals of 40 min, 80 min, 2 h, 3 h, 4 h, 5 h, 7 h, 9 h, 20 h. Among them, 100 μL was used to measure the release amount of RES at 306 nm; another 100 μL was used to measure the release amount of OPC at 500 nm. After each sampling, 200 μL of fresh samples (SGF, SIF and SCF) was added to the digestive fluid to keep the volume constant. The results are expressed as the percentage of cumulative release (CR). The cumulative release rates of RES and OPC were calculated using the formula.

[0082]

[0083] C n and C n-1 represent the concentrations (mg / mL) of the active substances in the release medium at the nth and (n - 1)th samplings respectively. n represents the sampling times, and L represents the amount (mg) of the active substances loaded in the hydrogel. The results are as Figure 5 shown.

[0084] The method for determining RES in the sample is as follows: First, draw a standard curve of RES. Weigh 0.1 g of RES and dissolve it with anhydrous ethanol solution with a concentration of 99.5% (mL / mL) to prepare a 1 mg / mL stock solution. Transfer 1 mL of the stock solution to a 10 mL brown volumetric flask, add distilled water to the mark, and shake well to obtain a RES solution with a concentration of 100 μg / mL. Respectively measure 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mL of the solution and place them in 10 mL volumetric flasks, add distilled water to the mark, and shake well to obtain different concentrations of RES solutions with concentrations of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 μg / mL for standby. Use a microplate reader to measure the absorbance values of the RES solutions with different concentrations at 306 nm, perform linear fitting of the concentration (μg / mL) against the absorbance value to obtain the RES standard curve. Substitute the absorbance value of the sample to be measured at 306 nm into the standard curve to obtain the corresponding release amount.

[0085] The method for determining proanthocyanidins in the sample is as follows: First, draw a standard curve of proanthocyanidins. Add 0.100 g of proanthocyanidin standard to a beaker, dissolve it with methanol and transfer it to a 100 mL volumetric flask, and make up the volume with methanol. Accurately measure 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, and 2.5 mL of the proanthocyanidin standard solution, add them to a 25 mL volumetric flask, and make up the volume with methanol. At this time, proanthocyanidin solutions with concentrations of 0.02, 0.04, 0.06, 0.08, 0.1 mg / mL are obtained. Measure 0.1 mL of each of the above 5 concentrations of the standard solution into a test tube, add 0.3 mL of 5% (g / g) vanillin solution and 0.1 mL of hydrochloric acid with a concentration of 40 g / L (w / v) to the test tube. Take 0.1 mL of methanol solution, add 0.3 mL of 5% (g / g) vanillin solution and 0.1 mL of hydrochloric acid with a concentration of 40 g / L as a blank control. Shake well, then perform light-shielding treatment, place it in a water bath, and react at 30 °C for 30 min. Measure the absorbance value of the above reacted solution with a microplate reader at a wavelength of 500 nm to draw the standard curve of proanthocyanidins. Then measure 0.1 mL of the sample to be measured into a test tube, add 0.3 mL of 5% (g / g) vanillin solution and 0.1 mL of hydrochloric acid with a concentration of 40 g / L (w / v) to the test tube. Take 0.1 mL of methanol solution, add 0.3 mL of 5% (g / g) vanillin solution and 0.1 mL of hydrochloric acid with a concentration of 40 g / L as a blank control. Shake well, then perform light-shielding treatment, place it in a water bath, and react at 30 °C for 30 min. Measure the absorbance value of the above reacted solution with a microplate reader at a wavelength of 500 nm, and then substitute it into the standard curve to obtain the release amount of proanthocyanidins.

[0086] Example 3:

[0087] 1. Preparation of R-SNPs-P-GEL.

[0088] (1) Dissolve 420 mg of shellac and 60 mg of resveratrol in 20 mL of anhydrous ethanol solution with a concentration of 99.5% (mL / mL).

[0089] (2) Filter out the insoluble substances in the solution of step (1) using a 0.22 μm microporous filter membrane.

[0090] (3) Introduce the filtrate obtained in step (2) into an HL-2B digital display constant current pump, and use a needle with an inner diameter of 0.27 mm to drop it into 90 mL of deionized water at a speed of 0.55 mL / min to obtain a dispersion of shellac alcohol nanoparticles loaded with resveratrol.

[0091] (4) Filter the dispersion obtained in step (3) with a slow filter paper, and use a rotary evaporator (R-1001VN, Zhengzhou Great Wall Scientific Industry and Trade Co., Ltd.) to evaporate and remove ethanol at 110 MPa, 52 °C, and 70 r / min to obtain a concentrated nano-dispersion.

[0092] (5) Add deionized water to the concentrated dispersion in step (4) until the total volume is 90 mL to obtain a dispersion of shellac nanoparticles.

[0093] (6) Take 80 mL of the solution in step (5) and dissolve 80 mg of procyanidins in this solution.

[0094] (7) Add 1.2 g of sodium alginate to the solution obtained in step (6) under mechanical stirring at 400 r / min to obtain a sol.

[0095] (8) Centrifuge at a speed of 1200 r / min for 15 min to remove the bubbles in the sol obtained in step (7).

[0096] (9) Drop the sol obtained in step (8) into 40 mL of 2% (g / mL) CaCl2 solution through a microfluidic device using a needle with an inner diameter of 0.3 mm at a speed of 2 mL / h to form a microgel.

[0097] (10) Let it stand for 30 min to solidify the microgel to obtain calcium alginate microgel.

[0098] 2. After adapting for one week, 50 male C57BL / 6 mice were randomly divided into 5 groups, with 10 mice in each group, which were respectively denoted as the Health (blank control) group, the Model (UC model) group, the 5-ASA (5-aminosalicylic acid positive control) group, the RES (R-SNPs gavage) group, and the RES+OPC (R-SNPs-P-GEL gavage) group. The breeding environment was clean grade, with a temperature of 23-24 °C, a humidity of 50-60%, a light / dark cycle (7:00-9:00), and free access to food and water. The animal care and use procedures were approved by the Ethics Committee of Hubei Provincial Center for Disease Control and Prevention and followed all applicable institutional and government regulations regarding the ethical use of animals. All mice were ensured free access to standard pellet feed during the 15-day experimental period. Among them, the Health group was ensured free drinking water during the 15-day experimental period, and the remaining 4 groups of mice were allowed to freely drink a 30 g / L dextran sulfate sodium salt (DSS) aqueous solution for the first 7 days, and then changed to free drinking water on the 8th day. Starting from the 4th day, the 5-ASA group, the RES group, and the RES+OPC group were continuously gavaged with 5-aminosalicylic acid solution (200 μL / kg / day), R-SNPs solution (200 μL / kg / day), and R-SNPs-P-GEL (30 mg / kg / day) respectively. Among them, 30 mg in R-SNPs-P-GEL (30 mg / kg / day) refers to the mass of R-SNPs-P-GEL, the mass of 5-aminosalicylic acid in 200 μL of 5-aminosalicylic acid solution is 2 mg, and the mass of R-SNPs in 200 μL of R-SNPs solution is 30 mg. The solution was prepared or diluted to ensure the dosage amount on the premise that the gavage volume was 200 μL.

[0099] (1) During the entire treatment period, the body weight of the mice was recorded daily, and a body weight change graph was drawn. The changes in body weight, visible stool consistency, and fecal bleeding were evaluated daily. The disease activity index (DAI) was determined by scoring the changes in body weight, stool viscosity, and bleeding. Among them, 0 points indicate normal feces, normal blood in feces, and no change in body weight; 1 point indicates soft stools sticking to the cage wall, with fewer blood cell spots in the feces, and a 0.1-5% weight loss; 2 points indicate moderate diarrhea with uneven stools, visible blood in the feces, and a 5.1-10% weight loss; 3 points indicate diarrhea (watery stools), fresh blood in the feces, and a 10.1-15% weight loss; 4 points indicate a weight loss >15%. The body weight change graph and DAI are as Figure 6As shown, it can be seen that the body weights of the mice in the Health group have been increasing for 15 days. The body weights of the mice in the Model group have been significantly decreasing since the 3rd day. On the 8th day, the body weights of the mice in the RES+OPC group gradually recovered. On the 12th day, the body weights of the mice in the 5-ASA group and the RES group also started to increase. Among them, the RES+OPC group had the most obvious increase effect, followed by the 5-ASA group and the RES group. This indicates that the interventions with 5-ASA, R-SNPs, and R-SNPs-P-GEL all have certain effects on alleviating UC. Among them, R-SNPs-P-GEL showed the best performance. The DAI of the mice treated with DSS started to increase from the 3rd day. From the 7th day, the increase in DAI of all intervention groups slowed down, showing a positive intervention effect. From the 8th day, the DAI of all DSS-treated mice started to show a downward trend. However, the DAI of the mice in the Model group was significantly greater than that of other groups, indicating that their UC symptoms were more obvious and the degree of colon inflammation was higher. The interventions with 5-ASA, R-SNPs, and R-SNPs-P-GEL can all alleviate the symptoms of UC to a certain extent.

[0100] (2) Twenty-four hours after the last administration, the mice were anesthetized with sodium pentobarbital and blood was collected. The mice in each group were sacrificed by cervical dislocation. The abdomen of the mice was quickly dissected, and the colon from 2 cm above the anus to 8 cm was completely removed. The colon length was measured, photographed and saved. Then, the intestinal cavity was cut along the longitudinal axis of the mesentery and quickly rinsed with ice-cold physiological saline to obtain the most severely damaged colon lesions. One part of them was fixed in 40 g / L formaldehyde, and the other part was immediately placed in liquid nitrogen and then stored in a -70 °C refrigerator. The changes in the obtained colon length are as Figure 7 , and it can be seen that compared with the Model control group, the colon lengths of the RES group and the RES+OPC group were significantly longer. This indicates that the interventions with R-SNPs and R-SNPs-P-GEL have potential application value in the prevention and treatment of UC. The preserved mouse colon tissues were subjected to paraffin embedding section experiments and hematoxylin-eosin staining histological evaluations. The obtained results are as Figure 8 , and it can be seen that the Health group showed normal tissue structure. In the Model group, irregularly shaped crypts were visible in the proximal part, while crypt abscesses and severe immune cell infiltration were visible in the distal part of the colon. All the epithelial cells in the mucosal layer in the field of view were eroded and shed, the lamina propria was exposed, and almost all the goblet cells and glandular structures in the mucosal layer disappeared, and the submucosa was edematous. In the RES+OPC group, there was an obvious improvement in inflammation. There were a large number of goblet cells in both the proximal and distal regions of the colon, with normal crypt structures, no crypt abscesses, and no accumulation of immune cells, being closer to the colon tissues of healthy mice.

[0101] (3) The spleens were collected, photographed, weighed, and the spleen index (the weight of the spleen relative to the body weight of the mouse) was calculated. The results are asFigure 9 As shown, the spleen of colitis mice was significantly enlarged, while the treatment with R-SNPs-P-GEL significantly reduced the spleen size. This indicates that R-SNPs-P-GEL has good effects on murine ulcerative colitis.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and 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 calcium alginate microgels for simultaneously delivering amphiphilic active substances, characterized in that It includes the following steps: (1) Dissolve shellac and resveratrol in an absolute ethanol solution; (2) Filter to remove the insoluble substances in the solution of step (1); (3) Drop the filtrate obtained in step (2) into water to obtain a dispersion of shellac-alcohol nanoparticles loaded with resveratrol; (4) Filter and evaporate the dispersion obtained in step (3) to obtain a concentrated dispersion with ethanol removed; (5) Add water to the concentrated dispersion in step (4) to obtain a dispersion of shellac nanoparticles; (6) Take the dispersion obtained in step (5) and dissolve proanthocyanidins in it; (7) Add sodium alginate to the solution obtained in step (6) under stirring conditions to obtain a sol; (8) Centrifuge to remove the bubbles in the sol obtained in step (7); (9) Drop the sol obtained in step (8) into a CaCl2 solution through a microfluidic device to form a microgel; (10) Let the microgel stand and solidify to obtain a calcium alginate microgel.

2. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The mass ratio of shellac to resveratrol in step (1) is 9:1 to 4:

1.

3. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The dosage of the absolute ethanol in step (1) satisfies that for every 60 mg of resveratrol, 20 - 45 mL of the absolute ethanol solution is added; The diameter of the microporous filter membrane used for filtration in step (2) is 0.22 - 0.45 μm; The dropping rate in step (3) is 0.3 - 0.6 mL / min, and the inner diameter of the needle used is 0.2 - 0.3 mm.

4. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The dosage of water added in step (5) satisfies that the concentration of the obtained dispersion of shellac nanoparticles is 3 - 6 g / L.

5. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The mass ratio of proanthocyanidins, shellac-alcohol nanoparticles loaded with resveratrol to sodium alginate in steps (6) and (7) is 1:3:7 to 1:10:

40.

6. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The stirring speed of the stirring in step (7) is 200 - 400 r / min; The centrifugation in step (8) means centrifuging at 1000 - 1500 r / min for 10 - 20 min.

7. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, wherein: The specific operation of dropping the sol into the CaCl2 solution through the microfluidic device in step (9) is: using an injection pump to drop the prepared sol into a 1.5 - 2.5% CaCl2 solution with a needle having an inner diameter of 0.25 - 0.35 mm at a speed of 2 - 5 mL / h.

8. The preparation method of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 1, characterized in that: In step (9), the dosages of the sol and the CaCl2 solution satisfy that the ratio of the mass of sodium alginate in the sol to the mass of CaCl2 is 1:5 to 1.5:1; The standing and curing time in the said step (10) is 20 to 40 min.

9. A calcium alginate microgel for simultaneously delivering amphiphilic active substances prepared by the method according to any one of claims 1-8.

10. The application of the calcium alginate microgel for simultaneously delivering amphiphilic active substances according to claim 9 in the preparation of drugs for relieving ulcerative colitis.

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