Complex gel at tail end of dopamine loaded with sinapine and application of complex gel
Through the dopamine-terminal composite gel loaded with mustardine, combined with the advantages of zein and polyethylene glycol, the problem of low bioavailability of mustardine is solved, achieving its long-term continuous release and high bioavailability in the intestine.
Patent Information
- Application Number
- CN202510193958.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-21
- Publication Date
- 2025-05-30
AI Technical Summary
Mustard is rapidly metabolized in the human gastrointestinal tract, resulting in low bioavailability, limiting its application as a functional food or drug ingredient. The existing polyphenol delivery system has a short residence time in the intestine, making it difficult to achieve long-term continuous release.
The composite gel with dopamine-terminal loading of mustardine is used to form a composite gel with intestinal adhesion and stability by combining the carrier advantages of zein, the hydrophilic advantages of polyethylene glycol chains and the adhesion advantages of dopamine, thereby achieving long-term and continuous release of mustardine.
It improves the encapsulation rate and intestinal residence time, achieves the long-term and continuous release of mustardine, enhances its bioavailability, and has excellent biocompatibility.
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Figure CN120052544A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of food processing, and particularly relates to a preparation method and application of an intestinal adhesion composite gel for delivering sinapine. Background Art
[0002] Sinapine is the most important polyphenolic compound in rapeseed, and has various biological activities such as antioxidant, anti-inflammatory and anti-tumor. Existing studies have confirmed that sinapine can prevent and improve liver damage. However, due to the rapid metabolism of sinapine in the human gastrointestinal tract, only a small amount can reach the liver, which results in its low bioavailability. This limitation hinders the wider application of sinapine as a functional food or drug ingredient.
[0003] Zein has good biodegradability and biocompatibility. It is the main storage protein of corn, has good self-loading property, is rich in hydrophobic amino acids such as proline, is insoluble in pure water, but soluble in ethanol-aqueous solution. Research reports that zein can form nanoparticles (NPs) by anti-solvent precipitation and can effectively encapsulate small molecule bioactive substances. However, the nanoparticles composed of single zein are unstable in weak acidic environment, lose stability in low salt ion and high temperature environment, the electrolyte components in gastrointestinal fluid will destroy its nanostructure, and the redispersibility of the powder obtained after freeze-drying or spray-drying is poor, with low oral bioavailability, which greatly limits its application in delivering bioactive substances. Summary of the Invention
[0004] Object of the Invention: To solve the above technical problems, the present invention provides a composite gel with dopamine-terminated sinapine loading and its application. The present invention solves the problems that sinapine is unstable and easily decomposed in an alkaline environment, and the existing polyphenol delivery system has a short residence time in the intestine, combines the carrier advantages of zein with the hydrophilic advantages of polyethylene glycol chain and the adhesion advantages of dopamine, follows the concept of green and safe development, and creates favorable conditions for the practical application of the oral delivery system of water-soluble drugs at the same time.
[0005] Technical Solution: To achieve the above object of the invention, the present invention adopts the following technical solution: A composite gel with dopamine-terminated sinapine loading is prepared by the following method: S1, Weigh anhydrous polyethylene glycol, melt it, add isocyanate and a catalyst, and react under nitrogen protection to obtain PEG with NCO end groups; S2, Dissolve an appropriate amount of anhydrous tetrahydrofuran in the PEG with NCO end groups obtained in step S1; S3. Continuously add the dimethylformamide solution of dopamine hydrochloride and anhydrous triethylamine into the system to form a hydrophilic polyethylene glycol shell modified with dopamine at the end. S4. Take the dimethylformamide solution of zein as the hydrophobic core of the composite, and add it to the hydrophilic polyethylene glycol shell obtained in step S3 to form a composite gel with dopamine at the end through particle assembly. S5. Continuously add the PBS solution of sinapine into the composite gel with dopamine at the end obtained in step S4, and vortex for more than 20 s to encapsulate sinapine, obtaining a composite gel with dopamine at the end loaded with sinapine.
[0006] Preferably, the isocyanate and the catalyst are isophorone diisocyanate and dibutyltin dilaurate respectively.
[0007] Preferably, in step S1, the addition amount of the isocyanate is 15% - 30% of the addition amount of polyethylene glycol, and the addition amount of the catalyst is 0.7% - 1.4% of the addition amount of the isocyanate.
[0008] Preferably, in step S3, the concentration of the dimethylformamide solution of dopamine hydrochloride is 10 mg / ml - 16 mg / ml (i.e., the mass ratio of dimethylformamide to dopamine hydrochloride is 62.5:1 - 100:1).
[0009] Preferably, in step S3, the addition amount of dopamine hydrochloride is 0.5% - 0.8% of the addition amount of anhydrous polyethylene glycol. Anhydrous triethylamine is used as an initiator, and its addition amount is 30% - 35% of the addition amount of dopamine hydrochloride.
[0010] Preferably, in step S4, the addition amount of zein is 3% - 5% of the addition amount of anhydrous polyethylene glycol.
[0011] Preferably, in step S5, the concentration of the PBS solution of sinapine is 8 μg / ml - 12 μg / ml, and the mass ratio of the addition amount of the PBS solution of sinapine to the composite gel with dopamine at the end is 1.25:1 - 3:1.
[0012] Preferably, the average molecular weight of the polyethylene glycol in step S1 is 1200 - 2000.
[0013] The present invention also provides an application of the above-mentioned composite gel with dopamine at the end loaded with sinapine for delivering sinapine to the human intestine and having intestinal adhesiveness.
[0014] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The operation process of the composite gel with intestinal adhesion property for delivering sinapine prepared by the present invention is simple, the amount of organic solvent used is small, and it has excellent biocompatibility. The composite gel with intestinal adhesion property for delivering sinapine prepared by the present invention has a hydrophobic protein core, which can increase the encapsulation rate of sinapine. In addition, the surface has a PEG shell modified with dopamine as the end group, which can enhance the adhesion of the composite, prolong the existence time of the composite in the intestine, and achieve the long-term sustained release of sinapine. Description of the Drawings
[0015] Figure 1 Comparative diagram of the maximum tensile force that the composite gels prepared in Example 1 and Comparative Examples 1-2 of the present invention can withstand; Figure 2 Morphology diagrams of the composite gels prepared in Example 1 and Comparative Examples 1-2 of the present invention after standing in different pH solutions for 2 days; Figure 3 Column chart of the protein content in the solution after in vitro simulated digestion of the composite gel prepared in Example 1 of the present invention; Figure 4 Fourier transform infrared spectra of the composite gels prepared in Steps S3 and S4 of Example 1 of the present invention and Comparative Examples 1-2; Figure 5 Rheological stress sweep diagrams of the composite gels prepared in Example 1 and Comparative Example 2 of the present invention; Figure 6 Flow morphology diagrams of Example 1 of the present invention on the intestinal wall over time; Figure 7 Column chart of the release rate of sinapine from the composite gel loaded with sinapine prepared in Example 1 of the present invention in simulated gastric and intestinal fluid environments. Detailed Embodiments
[0016] The following further clarifies the present invention in conjunction with the drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art fall within the scope defined by the appended claims of this application.
[0017] Raw materials in the embodiments of the present invention: Polyethylene glycol was purchased from the National Pharmaceutical Group; Sinapine was purchased from Chengdu Efar Biotechnology Co., Ltd.; Zein was from Shanghai Yuanye Co., Ltd.; Dopamine hydrochloride was purchased from Shanghai Macklin Biochemical Co., Ltd. Example
[0018] Step S1: Prepare polymerized PEG with NCO end groups Step 1: Melt 10 g of anhydrous polyethylene glycol with an average molecular weight of 1500 in a dry three-necked round-bottom flask.
[0019] Step 2: Add 1.5 mL of isophorone diisocyanate and 20 μL of dibutyltin dilaurate, and react at 80 °C for 60 min under the protection of nitrogen to generate NCO-capped polymeric PEG.
[0020] Step S2: Prepare a hydrophilic polyethylene glycol shell with dopamine-terminated end-group modification Step 1: Cool the NCO-capped polymeric PEG to room temperature, add an appropriate amount of anhydrous tetrahydrofuran to adjust the viscosity, and dissolve the above-mentioned polymerized polyethylene glycol.
[0021] Step 2: Add 80 mg of dopamine hydrochloride dissolved in 5 mL of anhydrous dimethylformamide to the system, and then add 26.7 mg of anhydrous triethylamine and react for 30 min to generate a hydrophilic polyethylene glycol shell with dopamine-terminated end-group modification.
[0022] Step S3: Prepare a composite gel with dopamine terminals Step 1: Dissolve 0.4 g of zein in 5 mL of anhydrous dimethylformamide as the hydrophobic core of the composite.
[0023] Step 2: Add the hydrophobic core in Step 1 to the hydrophilic polyethylene glycol shell and react for 1 h to form the final amphiphilic particles, so as to form a composite gel with dopamine terminals through particle assembly.
[0024] Step S4: Prepare a composite gel with dopamine terminals loaded with sinapine Step 1: Dissolve 100 μg of sinapine in 10 mL of PBS to prepare a 10 μg / mL sinapine-PBS solution.
[0025] Step 2: Take 1 mL of the 10 μg / mL sinapine-PBS solution and add it to 0.5 mL of the composite gel with dopamine terminals obtained in Example 3, and vortex for 30 s to encapsulate sinapine, obtaining a composite gel with dopamine terminals loaded with sinapine. Example
[0026] This example is different from Example 1 in that: the adhesion evaluation and acid-base resistance experiments are carried out on Example 1, and the specific steps are as follows: Step 1: Wash the fresh pig small intestine 3 times with PBS buffer solution, degrease it, select the part with uniform thickness, cut it into intestinal segments of the same size, flatten it and place it on ice for later use.
[0027] Step 2: Evenly apply the composite gel prepared in Example 1 to 1 / 2 of the inner wall area of the porcine small intestine in Step 1, and then quickly cover the side coated with the gel with another section of porcine small intestine. After standing for one hour, conduct a tensile test.
[0028] Step 3: Place the same volume of the composite gel in UP water, buffer solutions with pH = 2 and pH = 7.5 respectively, and let it stand for 2 days, then observe the changes in the composite gel.
[0029] Step 4: Prepare simulated gastric juice and simulated intestinal juice, and store them in a refrigerator at 4°C.
[0030] Step 5: Take the same volume of Example 1 and place it in simulated gastric juice and simulated intestinal juice respectively, and let it stand at 37°C for 1 h to conduct a simulated gastrointestinal digestion experiment, and then use the Kjeldahl method to measure the protein content in the solution. Example
[0031] Compared with Example 1, this example is different in that: infrared spectrum characterization and rheological scanning are performed on Example 1, and the specific steps are as follows: Step 1: Place Example 1 in a freeze dryer and freeze-dry it for three days, then take it out and perform infrared spectrum scanning using an infrared spectrometer in the wavenumber range of 7800 - 375 cm -1 Wavenumber range.
[0032] Step 2: Use a rotational rheometer, with a 20 mm, 1° cone, to perform strain scanning and time scanning on Example 1 and Comparative Example 2 at room temperature. At a constant frequency of 1 Hz, strain scanning is performed on Example 1 in the strain range of 0.1% - 100% to compare the rheological properties of Example 1 and Comparative Example 2. Since the composite gel prepared in Comparative Example 1 is not in a liquid state, rheological tests are not performed on it. Example
[0033] Compared with Example 1, this example is different in that: an in vitro adhesion test is performed on Example 1, and the specific steps are as follows: Step 1: Fresh intestinal tissues are purchased from the market and stored on ice. Before use, wash them three times with PBS buffer (1×), defat them, select parts with the same thickness, cut them into intestinal segments of the same size, and flatten them and place them on ice for standby.
[0034] Step 2: After staining the composite gel prepared in Example 1 with FCF, deposit it on the surface of the upright and moist intestinal mucosa. Take pictures of the composite gel at 0 s, 100 s, 200 s, and 300 s respectively, and record the flow state of the composite gel on the intestinal mucosa surface over time. Example
[0035] This example is different from Example 1 in that an in vitro sinapine release experiment was carried out on Example 1, and the specific steps are as follows: Step 1: Prepare simulated gastric juice and simulated intestinal juice, and store them in a refrigerator at 4°C.
[0036] Step 2: Take the same volume of the composite gel prepared in Example 1 and place it in the simulated gastric juice and intestinal juice prepared in Step 1, and incubate it in a water bath at 37°C.
[0037] Step 3: Take the supernatant at 30 minutes and 60 minutes of incubation respectively, centrifuge it, and use an ultraviolet spectrophotometer to measure the change in the concentration of sinapine in the supernatant at 326 nm.
[0038] Comparative Example 1 (Preparation of Composite Gel without Terminal) The preparation method of the composite gel without terminal in this comparative example is as follows: Step 1: Melt 10 g of anhydrous polyethylene glycol with an average molecular weight of 1500 in a dry three-necked round-bottom flask, add 1.5 mL of isophorone diisocyanate and 20 μL of dibutyltin dilaurate, and react at 80°C for 60 min under the protection of nitrogen to generate polymerized PEG with NCO end groups; Step 2: Cool the polymerized PEG with NCO end groups to room temperature, add an appropriate amount of anhydrous tetrahydrofuran to adjust the viscosity, and dissolve the above polymerized polyethylene glycol; Step 3: Add 5 mL of dimethylformamide to the system, then add 26.7 mg of anhydrous triethylamine and react for 30 min. The unmodified polymerized PEG with capped isocyanate (NCO) groups is directly used as the hydrophilic PEG shell of the composite gel.
[0039] Step 4: Dissolve 0.4 g of zein in 5 mL of anhydrous dimethylformamide as the hydrophobic core of the composite, and add it to the hydrophilic polyethylene glycol shell and react for 1 h to obtain a composite gel without terminal through particle assembly.
[0040] Comparative Example 2 (Preparation of Composite Gel with Methyl Terminal) The preparation method of the composite gel with methyl terminal in this comparative example is as follows: Step 1: Melt 10 g of anhydrous polyethylene glycol with an average molecular weight of 1500 in a dry three-necked round-bottom flask, add 1.5 mL of isophorone diisocyanate and 20 μL of dibutyltin dilaurate, and react at 80°C for 60 min under the protection of nitrogen to generate polymerized PEG with NCO end groups; Step 2: Cool the polymerized PEG with NCO end groups to room temperature, add an appropriate amount of anhydrous tetrahydrofuran to adjust the viscosity, and dissolve the above polymerized polyethylene glycol; Step 3: 5 mL of toluene was added to the system, followed by 26.7 mg of anhydrous triethylamine and reacted for 30 min to generate a hydrophilic polyethylene glycol shell with a methyl-terminated end group; Step 4: 0.4 g of zein was dissolved in 5 mL of anhydrous dimethylformamide as the hydrophobic core of the complex, added to the hydrophilic polyethylene glycol shell, and reacted for 1 h to obtain a composite gel with methyl termini through particle assembly.
[0041] like Figure 1-7 As shown, in order to evaluate the wet adhesion effect of Example 1, we applied Example 1, Comparative Example 1 and Comparative Example 2 to the inner wall of pig small intestine and carried out tensile tests. The small intestine tissue adhered by the composite gel of Example 1 can withstand an external force 3 times greater than that of Comparative Examples 1 and 2.
[0042] In order to evaluate whether the composite gel of Example 1 has good acid and alkali resistance, Example 1, Comparative Example 1 and Comparative Example 2 were injected into pH=2, pH=7.4 and UP water respectively for two days, and the changes of the three composite gels in the solution were observed. It can be clearly seen that Example 1 remains stable in a buffer solution with a wide pH range, while the control groups have different degrees of dispersion, among which the phenomenon of Comparative Example 2 is the most obvious. It can be observed that zein is precipitated on the surface of the buffer solution, and the solution is white as a whole.
[0043] The results of in vitro simulated gastrointestinal digestion showed that the protein contents in simulated gastric juice and intestinal juice were 0.91% and 0.59%, respectively, indicating that the complex can remain stable in acidic and weakly alkaline environments.
[0044] The FTIR spectra of free sinapinic acid and the composite gel prepared in steps S3 and S4 of Example 1, Comparative Example 1 and Comparative Example 2 are as follows: Figure 3 As shown. Compared with free mustard, the composite gel loaded with mustard prepared in Example 1 and the composite gel with dopamine ends prepared in Example 1 have no absorption peak at 2059.6 cm-1, indicating that mustard is successfully embedded in the zein core of the composite gel. The four composite gels show sharp and strong peaks near 2800 cm-1 and 1010 cm-1, corresponding to the CH stretching vibration of saturated hydrocarbons and the in-plane bending vibration of CH, respectively. Here, the peak intensity of Comparative Example 1 is weaker than that of the other three groups, because the composite has no end group. The composite gels prepared in steps S3 and S4 of Example 1 show hydroxyl stretching bands at 3567.7 cm-1 and 3531.0 cm-1, respectively, corresponding to amide A bands (hydrogen bonds and NH stretching), proving the presence of hydrogen bonds in these two types of composite gels.
[0045] G’ refers to the storage modulus, representing the elastic part of the viscoelastic behavior. G” refers to the loss modulus, describing the viscous part of the viscoelastic behavior. As the strain varies in the range of 0.01 % - 100 %, the loss modulus G” of the two composite gels is greater than the storage modulus G’, and within this range, G” (Example 1) > G” (Comparative Example 2), indicating that both composite gels have excellent fluidity and adhesiveness. In the strain range less than 1 %, G’ (Example 1) > G’ (Comparative Example 2), and when the strain is 0.3%, G’ (Example 1) decreases significantly, showing shear thinning phenomenon and poor fluidity. While G’ (Comparative Example 2) rises sharply, showing shear thickening phenomenon and enhanced fluidity, and then tends to be stable.
[0046] After dyeing Example 1, it was deposited on the inner wall of the vertical small intestine tissue. Due to its good adhesiveness and fluidity, it can be observed that it can adhere to the surface of the fresh and moist small intestine mucosa, and over time, it flows steadily downward to cover the intestinal mucosa surface to form a large-area adhesion coating.
[0047] In vitro release experiments showed that sinapine had different release rates in simulated gastric juice and intestinal juice. In simulated gastric juice, the release rates of sinapine at 30 min and 60 min were 0.15% and 0.26% respectively, and in simulated intestinal juice, the release rates were 0.68% and 0.69% respectively. The same is that there is only a slight increase. And the release in simulated intestinal juice is more than that in gastric juice, indicating that Example 1 can not only achieve the sustained release of water-soluble drugs but also achieve the controlled release in the gastrointestinal tract.
[0048] The zein selected in the present invention has good biodegradability and biocompatibility and is a recognized food safety material. It carries a positive charge, and the content of non-polar amino acids (including proline, alanine, and alanine) is greater than 50%. Assembling hydrophilic or amphiphilic substances on the surface of zein nanoparticles can effectively improve its oral bioavailability by enhancing its stability to pH or salt ions. Polyethylene glycol (PEG) is a common hydrophilic polymer for forming a shell, which can self-assemble with polypeptides to form core-shell micelles. PEGylation of zein can prevent opsonization and provide steric hindrance for the enzymatic degradation of zein. Dopamine (DA) is a water-soluble small molecule substance found in marine mussel mucus and can be polymerized onto PEG as a terminal group. Compared with the composite gel without a terminal and the composite gel with a methyl terminal, the composite gel with a dopamine terminal has pH independence (see Example 2 in detail), endowing the composite gel with good stability, and can form a large-area and uniform adhesion coating on the intestine to mediate the sustained release of sinapine.
[0049] The above are only some embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A composite gel loaded with dopamine ends of sinapinic acid, characterized in that: Prepared by the following method: S1, weighing anhydrous polyethylene glycol and melting it, adding isocyanate and a catalyst, and reacting under nitrogen protection to obtain PEG with NCO end-capping; S2, adding an appropriate amount of anhydrous tetrahydrofuran to dissolve the NCO-terminated PEG obtained in step S1; S3, continuing to add a dimethylformamide solution of dopamine hydrochloride and anhydrous triethylamine into the system to generate a hydrophilic polyethylene glycol shell with dopamine terminal modification; S4, adding a dimethylformamide solution of zein as a hydrophobic core of the complex to the hydrophilic polyethylene glycol shell obtained in step S3, and forming a composite gel with dopamine ends through particle assembly; S5, continuing to add the PBS solution of sinapinic acid to the composite gel with dopamine terminals obtained in step S4, and vortexing for more than 20 seconds to encapsulate sinapinic acid, thereby obtaining a composite gel with dopamine terminals loaded with sinapinic acid.
2. The composite gel of dopamine terminal loaded with sinapinic acid according to claim 1, characterized in that: The isocyanate and the catalyst are isophorone diisocyanate and dibutyltin dilaurate respectively.
3. The composite gel of dopamine terminal loaded with sinapinic acid according to claim 2, characterized in that: In step S1, the amount of isocyanate added is 15%-30% of the amount of polyethylene glycol added, and the amount of catalyst added is 0.7%-1.4% of the amount of isocyanate added.
4. The composite gel of dopamine terminal loaded with sinapinic acid according to claim 1, characterized in that: In step S3, the concentration of the dimethylformamide solution of dopamine hydrochloride is 10 mg / ml-16 mg / ml (ie, the mass ratio of dimethylformamide to dopamine hydrochloride is 62.5:1-100:1).
5. The composite gel of dopamine terminal loaded with sinapinic acid according to claim 1, characterized in that: In step S3, the amount of dopamine hydrochloride added is 0.5%-0.8% of the amount of anhydrous polyethylene glycol added, and the amount of anhydrous triethylamine as an initiator is 30%-35% of the amount of dopamine hydrochloride added.
6. The composite gel of dopamine terminal loaded with sinapinic acid according to claim 1, characterized in that: In step S4, the amount of zein added is 3%-5% of the amount of anhydrous polyethylene glycol added.
7. The composite gel of dopamine terminals loaded with sinapinic acid according to claim 6, characterized in that: In step S5, the concentration of the PBS solution of sinapinic acid is 8 μg / ml-12 μg / ml, and the mass ratio of the added amount of the PBS solution of sinapinic acid to the composite gel having dopamine ends is 1.25:1-3:
1.
8. The composite gel of dopamine terminals loaded with sinapinic acid according to claim 1, characterized in that: The average molecular weight of the polyethylene glycol in step S1 is 1200-2000.
9. The dopamine-terminated composite gel loaded with sinapinic acid according to any one of claims 1 to 8, for use in delivering sinapinic acid to the human intestine and having intestinal adhesion.