Colon-targeted composite gel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of composite gel and application thereof, and relates to colon-targeted composite gel and a preparation method and application thereof.The preparation method of the colon-targeted composite gel comprises the following steps that S1, polysaccharide and protein are dissolved in water with the same volume, and a polysaccharide solution and a protein solution are correspondingly obtained; s2, mixing the protein solution and the polysaccharide solution, adding succinic acid and calcium chloride, and finally staying overnight at 4 DEG C to obtain the colon-targeted composite gel. Polysaccharide, protein, succinic acid and calcium chloride are adopted to form the colon-targeted composite gel, the colon-targeted composite gel has a compact and stable structure, the encapsulation efficiency of succinic acid is greatly improved, the targeted release rate of succinic acid in colon is facilitated, and precise targeted delivery of succinic acid in colon is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of composite gel and its application, and relates to a colon-targeted composite gel and a preparation method and application thereof. Background Art

[0002] With the rapid development of science and technology and the changes in people's dietary habits, obesity and glucose metabolism dysfunction have become increasingly serious worldwide in recent years, and the number of patients has shown an increasing trend year by year. At present, the treatment of diabetes mainly relies on oral hypoglycemic drugs and insulin injections, and in terms of preventive measures, in addition to paying attention to dietary adjustments, there is currently a lack of other more effective means to prevent the occurrence of diabetes.

[0003] Recent studies have found that the intestine has the ability of gluconeogenesis (IGN), and can negatively feedback regulate blood sugar disorders in the body through the "gut-brain-liver" axis, achieving the effect of controlling appetite and regulating blood sugar levels. Therefore, intestinal gluconeogenesis (IGN) is an important way for the body to regulate sugar metabolism. In the IGN pathway, succinic acid (SA) is a key substance that activates and participates in IGN, and studies have found that SA can be produced by the intestinal flora of the colon through the metabolism of dietary fiber. However, excessive intake of dietary fiber can easily cause indigestion, increase the burden on the gastrointestinal tract, and cause symptoms such as abdominal distension; and if small molecule carboxylic acid SA is taken orally, SA is easily absorbed by the small intestine into the systemic circulation, which may induce adverse reactions such as tissue and organ damage and inflammation, and it is difficult to reach the colon and activate IGN. Therefore, it is very critical and important to accurately deliver SA to the colon, activate IGN and regulate blood sugar homeostasis.

[0004] There is an existing method for delivering a target object to the colon. With reference to the Chinese patent document CN118109536A, a method for preparing a nano-resistant pure amylose barley starch is disclosed, comprising enzymatically hydrolyzing the pure amylose barley starch with a buffer solution containing barley germination powder, and then heating to obtain resistant pure amylose barley starch; adding ethanol to the resistant pure amylose barley starch, centrifuging, and drying to obtain nano-resistant pure amylose barley starch; adding a solution containing a fat-soluble stabilizer to an aqueous solution of the nano-resistant pure amylose barley starch, and freeze-drying to obtain a nano-resistant pure amylose barley starch stable complex; using an aqueous solution containing the nano-resistant pure amylose barley starch stable complex as an aqueous phase and an oil phase containing a fat-soluble drug through shearing to obtain a Pickering emulsion, wherein the nano-resistant starch can resist enzymatic hydrolysis in the stomach and small intestine, reach the colon and then be degraded, has anti-gastrointestinal digestion properties, and realizes emulsion targeted colon release.

[0005] However, the existing methods have a low encapsulation rate due to poor stability when encapsulating succinic acid, and cannot achieve precise targeted delivery of succinic acid in the colon, resulting in a low release rate. Summary of the invention

[0006] In order to solve the technical problem in the prior art that poor stability leads to low encapsulation rate and accurate targeted delivery of succinic acid in the colon cannot be achieved, the present invention provides a colon-targeted composite gel and a preparation method and application thereof.

[0007] The present invention adopts polysaccharide, protein, succinic acid and calcium chloride to form a colon-targeted composite gel, which has a dense and stable structure, greatly improves the encapsulation rate of succinic acid, is beneficial to the targeted release rate of succinic acid in the colon, and realizes the precise targeted delivery of succinic acid in the colon.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for preparing a colon-targeted composite gel comprises the following steps:

[0010] S1. Dissolve the polysaccharide and the protein in equal volumes of water to obtain a polysaccharide solution and a protein solution respectively; the mass ratio of the polysaccharide to the protein is (1.9-2.1):1;

[0011] S2. Mix the protein solution and the polysaccharide solution, then add succinic acid and calcium chloride, add 0.05wt%-0.1wt% succinic acid and 6wt%-8wt% calcium chloride to every 10ml of protein solution; finally, incubate at 4±0.5°C overnight to obtain a colon-targeted composite gel.

[0012] It is further defined that the polysaccharide is artemisia seed gum polysaccharide, low-ester pectin, chitosan or inulin.

[0013] In a further embodiment, the protein is lysozyme, glutaminase or whey protein isolate.

[0014] The colon-targeted composite gel is prepared by the preparation method of the colon-targeted composite gel.

[0015] It is further defined that the encapsulation rate of succinic acid in the colon-targeted composite gel reaches 87.75% to 98.92%.

[0016] The colon targeting composite gel is used in preparing medicine for treating colitis.

[0017] The colon-targeted composite gel is used in preparing drugs for treating colitis by improving the targeted release rate of succinic acid.

[0018] The colon-targeted composite gel is used in the preparation of a drug for treating colitis by enhancing the mRNA expression of genes.

[0019] It is further defined that the gene is G6Pase, PEPCK, SREBP-1c and / or Fas.

[0020] The colon-targeted composite gel is used in preparing a drug for treating colitis by improving the Shannon index and Chao index of type 2 diabetes model mice.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention adopts polysaccharide, protein and calcium chloride to form a cross-linking system to encapsulate succinic acid to form a colon-targeted composite gel, which has a dense and stable structure, greatly improves the encapsulation rate of succinic acid, is beneficial to the targeted release rate of succinic acid in the colon, and realizes the precise targeted delivery of succinic acid in the colon.

[0023] 2. In the present invention, cross-linking of polysaccharides and proteins can promote interaction and further enhance the interaction with Ca 2+ The network-like eggshell structure formed after cross-linking ensures that the composite gel structure is denser and enhances the thermal stability of the composite gel. Experiments have shown that the encapsulation rate of succinic acid in the composite gel is as high as 98.92%. The composite gel releases a large amount of SA after 30 minutes, reducing its premature release in the stomach and small intestine. It can deliver the target substance succinic acid precisely to the colon, thereby increasing the release rate of succinic acid in the colon.

[0024] 3. In the present invention, the colon-targeted composite gel, when used in the preparation of drugs for treating colitis, reduced the blood sugar level, glucose tolerance and insulin sensitivity of T2DM mice, and significantly increased the mRNA expression of gluconeogenesis-related genes G6Pase, PEPCK, SREBP-1c and Fas in the colon tissue of T2DM mice (p < 0.05), among which the mRNA expression of G6Pase, PEPCK and Fas genes was significantly increased compared with that of the NC group. At the same time, the IGN of T2DM mice was activated, which has the ability to regulate blood sugar.

[0025] 5. In the present invention, the colon-targeted composite gel can significantly increase the Shannon index and Chao index of T2DM mice (p < 0.05) in the preparation of drugs for treating colitis, and improve the diversity of intestinal flora in T2DM mice.

[0026] 6. The colon-targeted composite gel of the present invention mainly uses polysaccharides and proteins as raw materials, and pectin polysaccharide is selected, which has good safety, can meet food grade requirements, and can be used in the preparation of drugs for treating colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the particle size of four polysaccharide / protein composite gels containing calcium ions;

[0028] Figure 2 The microscopic morphologies of several different polysaccharide / protein composite gels;

[0029] Figure 3 The infrared spectra of four polysaccharide / protein composite gels containing calcium ions;

[0030] Figure 4 The XRD of four polysaccharide / protein composite gels containing calcium ions;

[0031] Figure 5 The DSC analysis results of four polysaccharide / protein composite gels containing calcium ions;

[0032] Figure 6 is the SA release rate of four colon-targeted composite gels loaded with succinic acid, Figure 6 A is the SA release rate in SGF(a), Figure 6 B is the SA release rate in SIF (b), Figure 6 C is the SA release rate in SCF (c);

[0033] Figure 7 To compare the weight results of each group;

[0034] Figure 8 To compare the liver and kidney results among the groups;

[0035] Fig. 9 The results of serum index comparison among the groups are shown in Figure 2.

[0036] Fig.10 To compare the analysis results of blood sample indicators in each group;

[0037] Fig.11 PC1 results of principal coordinate analysis of intestinal flora;

[0038] Fig.12 This is the hierarchical clustering result of intestinal flora;

[0039] Fig.13 The Kruskal-Wallis test plots of the intestinal flora of each group;

[0040] Fig.14 The bar charts of intestinal flora analysis of each group;

[0041] Fig.15 Results for the intestinal flora of mice;

[0042] Fig.16 The Shannon index and Chao index results of each group;

[0043] Fig.17 The test results of short-chain fatty acids in each group;

[0044] Fig.18 The results of analysis of succinate content in the colon of each group;

[0045] Fig.19The results of analysis of succinate content in the cecum of each group;

[0046] Fig. 20 are the analysis results of succinate content in the small intestine of each group;

[0047] Fig.21 These are the mRNA expression results of gluconeogenesis-related genes G6Pase, PEPCK, SREBP-1c and Fas. DETAILED DESCRIPTION

[0048] The technical solution protected by the present invention is now described in detail in conjunction with the accompanying drawings and embodiments. However, it is obvious that the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present application.

[0049] The present invention provides a method for preparing a colon-targeted composite gel, comprising the following steps:

[0050] S1. Dissolve the polysaccharide and the protein in equal volumes of water to obtain a polysaccharide solution and a protein solution respectively; the mass ratio of the polysaccharide to the protein is (1.9-2.1):1;

[0051] S2. Mix the protein solution and the polysaccharide solution, then add succinic acid and calcium chloride, add 0.05wt%-0.1wt% succinic acid and 6wt%-8wt% calcium chloride to every 10ml of protein solution; finally, incubate at 4±0.5°C overnight to obtain a colon-targeted composite gel.

[0052] Preferably, the polysaccharide is artemisia seed gum polysaccharide, low-ester pectin, chitosan or inulin.

[0053] Preferably, the protein is lysozyme, glutaminase or whey protein isolate.

[0054] The colon-targeted composite gel obtained by the preparation method of the present invention has an encapsulation rate of succinic acid of 87.75% to 98.92%. In the preparation of drugs for treating colitis, the targeted release rate of succinic acid in the colon can be improved, the mRNA expression of genes can be increased, and the Shannon index and Chao index of type 2 diabetes model mice can be improved.

[0055] The technical solution and performance of the present invention are described below through several groups of specific embodiments.

[0056] The materials and equipment used in the following examples are as follows.

[0057] 1. Materials and Reagents

[0058] Low ester pectin (LEP, degree of esterification ≤ 50%) was extracted from apple and purchased from Shandong Qilu Biotechnology Co., Ltd. Artemisia sphaerocephala seed polysaccharide (SSG) was purchased from Hubei Gumu Biotechnology Co., Ltd. Calcium chloride (CaCl2), simulated gastric juice (containing pepsin), simulated small intestinal juice (containing trypsin and phosphate), simulated colonic juice, transglutaminase (TGase), lysozyme, succinic acid (SA) and other materials were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Conventional chemical reagents were all analytically pure reagents.

[0059] 2. Main instruments

[0060] In the following examples, the instruments used are shown in Table 1.

[0061] Table 1 Experimental instruments

[0062] Instrument Name model factory Fully automatic upright fluorescence microscope Axio Imager.M2 Carl Zeiss Constant temperature shaker QYC-2102C Taicang Huamei Biochemical Instrument Factory microscope CKX41 Olympus GC-MS instrument 2010ultra Japan Shimadzu Corporation Ultra-micro-spectrophotometer NanoDrop One Thermo Fisher Scientific Stable blood glucose meter Stable Sinocare Biosensing Co., Ltd. PCR instrument CFX Connect Optics Module Bio-rad ELISA reader HM-SY96S Hengmei Fourier transform infrared spectroscopy INVENNIO S BRUKER DSC Microscope DSC1 Professional Mettler Toledo X-ray diffractometer D8 DISCOVER A25 BRUKER Field Emission Scanning Electron Microscope Nava NanoSEM 450 Thermo Fisher Scientific

[0063] Example 1

[0064] The preparation method of the colon targeting composite gel provided in this embodiment comprises the following steps:

[0065] S1. Dissolve polysaccharide and protein in equal volumes of water to obtain polysaccharide solution and protein solution respectively.

[0066] 2 g of Artemisia sphaerocephala seed gum polysaccharide SSG was accurately weighed and dissolved in 10 mL of deionized water, and the mixture was stirred until it was completely dissolved to obtain a polysaccharide solution.

[0067] Weigh 1 g of lysozyme and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0068] S2. Mix the protein solution and polysaccharide solution, then add succinate SA and calcium chloride CaCl 2 , 0.05 g succinic acid and 0.8 g calcium chloride were added to every 10 ml protein solution; finally, the mixture was incubated at 4°C overnight to obtain a colon-targeted composite gel, i.e., a polysaccharide / protein composite gel loaded with SA, denoted as SA-loaded SSG / lysozyme / Ca 2+ Composite gel.

[0069] Example 2

[0070] The preparation method of the colon targeting composite gel provided in this embodiment comprises the following steps:

[0071] S1. Dissolve polysaccharide and protein in equal volumes of water to obtain polysaccharide solution and protein solution respectively.

[0072] 2 g of Artemisia sphaerocephala seed gum polysaccharide SSG was accurately weighed and dissolved in 10 mL of deionized water, and the mixture was stirred until it was completely dissolved to obtain a polysaccharide solution.

[0073] Weigh 1 g of transglutaminase TGase and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0074] S2. Mix the protein solution and polysaccharide solution, then add succinate SA and calcium chloride CaCl 2 , 0.05 g succinic acid and 0.8 g calcium chloride were added to each 10 ml protein solution; finally, the mixture was incubated at 4°C overnight to obtain a colon-targeted composite gel, i.e., a polysaccharide / protein composite gel loaded with SA, denoted as SA-loaded SSG / TGase / Ca 2+ Composite gel.

[0075] Example 3

[0076] The preparation method of the colon targeting composite gel provided in this embodiment comprises the following steps:

[0077] S1. Dissolve polysaccharide and protein in equal volumes of water to obtain polysaccharide solution and protein solution respectively.

[0078] 2 g of low-ester pectin LEP was accurately weighed and dissolved in 10 mL of deionized water, and the mixture was stirred until it was completely dissolved to obtain a polysaccharide solution.

[0079] Weigh 1 g of lysozyme and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0080] S2. Mix the protein solution and polysaccharide solution, then add succinate SA and calcium chloride CaCl 2 0.05 g succinic acid and 0.8 g calcium chloride were added to each 10 ml protein solution; finally, the mixture was incubated at 4°C overnight to obtain a colon-targeted composite gel, i.e., a polysaccharide / protein composite gel loaded with SA, which was recorded as SA-loaded LEP / lysozyme / Ca 2+ Composite gel.

[0081] Example 4

[0082] The preparation method of the colon targeting composite gel provided in this embodiment comprises the following steps:

[0083] S1. Dissolve polysaccharide and protein in equal volumes of water to obtain polysaccharide solution and protein solution respectively.

[0084] 2 g of low-ester pectin LEP was accurately weighed and dissolved in 10 mL of deionized water, and the mixture was stirred until it was completely dissolved to obtain a polysaccharide solution.

[0085] Weigh 1 g of transglutaminase TGase and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0086] S2. Mix the protein solution and polysaccharide solution, then add succinate SA and calcium chloride CaCl 2 , 0.05 g succinic acid and 0.8 g calcium chloride were added to each 10 ml protein solution; finally, the mixture was incubated at 4°C overnight to obtain a colon-targeted composite gel, i.e., a polysaccharide / protein composite gel loaded with SA, which was recorded as SA-loaded LEP / TGase / Ca 2+ Composite gel.

[0087] In order to verify the effect of the colon-targeted composite gel of the present invention on colon-targeted delivery, the composite gels prepared in Examples 1 to 4 were used to conduct the following experiments.

[0088] In order to study the structure and performance of the colon-targeted composite gel loaded with succinic acid of the present invention, the following polysaccharide / protein composite gel samples were prepared for comparison in the experiment.

[0089] Comparative Example 1

[0090] This comparative example provides a first method for preparing a polysaccharide / protein composite gel sample, comprising the following steps:

[0091] (1) Accurately weigh 2 g of polysaccharide and dissolve it in 10 mL of deionized water, and stir the mixture until it is completely dissolved to obtain a polysaccharide solution;

[0092] (2) Weigh 1 g of protein and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0093] (3) The protein solution was heated in a water bath at 85°C for 25 min. After heating, it was allowed to stand until it reached room temperature, and then 0.8 g of CaCl was added. 2 and fully stirred until completely dissolved to obtain a mixed solution;

[0094] (4) The polysaccharide solution and the mixed solution were mixed and placed in a refrigerator at 4°C overnight to obtain a protein / polysaccharide composite gel.

[0095] During preparation, the polysaccharide is selected from artemisia seed gum polysaccharide SSG or low-ester pectin LEP, and the protein is selected from lysozyme lysozyme or transglutaminase TGase.

[0096] According to the above method, SSG / lysozyme / Ca 2+ Composite gel, SSG / TGase / Ca 2+ Composite gel, LEP / lysozyme / Ca2+ Composite gel and LEP / TGase / Ca 2+ Composite gel.

[0097] Comparative Example 2

[0098] This comparative example provides a second method for preparing a polysaccharide / protein composite gel sample, comprising the following steps:

[0099] (1) Accurately weigh 2 g of polysaccharide and dissolve it in 10 mL of deionized water, and stir the mixture until it is completely dissolved to obtain a polysaccharide solution;

[0100] (2) Weigh 1 g of protein and dissolve it in 10 mL of deionized water. Stir thoroughly until it is completely dissolved to obtain a protein solution.

[0101] (3) The protein solution was heated in a water bath at 85°C for 25 min. After the heating was completed, the solution was allowed to stand until it reached room temperature to obtain a mixed solution.

[0102] (4) The polysaccharide solution and the mixed solution were mixed and placed in a refrigerator at 4°C overnight to obtain a protein / polysaccharide composite gel.

[0103] During the preparation, the polysaccharide is selected from artemisia seed gum polysaccharide SSG or low-ester pectin LEP, and the protein is selected from lysozyme lysozyme. According to the above method, SSG / lysozyme composite gel and LEP / lysozyme / composite gel are prepared respectively.

[0104] The properties of the composite gel materials prepared in this embodiment and the comparative example are compared and analyzed by physical property tests and chemical property tests.

[0105] Test 1: Determination of composite gel particle size

[0106] Sample: Take composite gel SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+ 、LEP / lysozyme / Ca 2+ and LEP / TGase / Ca 2+ ; Low ester pectin LEP and Artemisia sphaerocephala seed gum polysaccharide SSG.

[0107] Test method: After diluting the sample 200 times with distilled water, the average particle size of the sample was measured using a laser particle size analyzer (NanoBrook 90PlusPALS, Brookhaven Instruments, USA), and the measurement was repeated three times. Figure 1In the figure above: SSG / lysozyme, SSG / TGase, LEP / lysozyme and LEP / TGase refer to composite gels containing calcium ions.

[0108] See also Figure 1 The average particle size of the four cross-linked composite gels was significantly larger than that of LEP and SSG (p < 0.05). This is because the composition of the protein-polysaccharide composite gel has undergone a transformation from two single substances to a mixture; on the other hand, the protein was preheated (85°C) during the preparation of the composite gel, which allowed the protein molecules to unfold and fully expose the hydrophobic groups, and swell and aggregate to form protein aggregates under weak interaction forces such as hydrophobic bonds and disulfide bonds, and cross-link with polysaccharides. The whole process has undergone changes in the conformation of proteins in the composite particles, affecting their cross-linking with LEP and SSG, and forming a complex with a larger particle size than a single protein or polysaccharide.

[0109] Experiment 2: Scanning Electron Microscopy

[0110] Sample: Take SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+ 、LEP / lysozyme / Ca 2+ 、LEP / TGase / Ca 2 + , SSG / lysozyme and LEP / lysozyme six kinds of composite gels; low-ester pectin LEP, Artemisia seed gum polysaccharide SSG, transglutaminase TGase and lysozyme lysozyme.

[0111] Test method: A field emission scanning electron microscope (FEI, Nava Nano SEM 450) was used to observe the microstructure of each freeze-dried gel sample. The freeze-dried gel sample was fixed on a conductive double-sided adhesive, sprayed with a platinum coating (30mA, 45s), and observed at 10KV. The results are as follows: Figure 2 shown.

[0112] See also Figure 2 It can be seen that the two polysaccharides (LEP and SSG) without cross-linked proteins are lamellar and have a loose structure. However, the lamellar structure of the gel after adding protein cross-linking disappears and is replaced by a smoother and denser structure, which is most obvious after adding TGase. 2+ Composite cross-linking can further promote the flatness, smoothness and density of the gel, forming a composite gel with a uniform gel network and fewer pores.

[0113] Experiment 3: Fourier transform infrared spectroscopy

[0114] Sample: Take composite gel SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+ 、LEP / lysozyme / Ca 2+ and LEP / TGase / Ca 2+ ; Low ester pectin LEP and Artemisia sphaerocephala seed gum polysaccharide SSG.

[0115] Test method: Fourier transform infrared spectroscopy (FTIR, Invennio S, Brooker, Germany) was used to determine the information of molecular interactions in proteoglycan gel. FT-IR is a commonly used instrument for analyzing the interaction forces in substances. It can clearly reflect the functional groups of the sample and the interaction force information in the substance by measuring the absorption degree of the sample to infrared radiation of various wavelengths. It has the advantages of being simple, rapid, non-destructive, efficient, and green.

[0116] Specifically, the KBr tablet method was used to accurately weigh 3 mg of freeze-dried gel sample and mix it with 197 mg of KBr, grind it into fine powder and press it into a complete tablet. The wave number range was set to 4000 cm -1 -400cm -1 , resolution 4cm -1 , perform automatic scanning (64 times), and subtract the background value from the results. Each sample was measured three times in parallel, and the best peak shape was selected for analysis. The results are shown in Figure 3 shown.

[0117] See also Figure 3 , 3100 -1 -3500cm -1 The band is the stretching vibration of hydroxyl group, and the characteristic peak of -OH in LEP appears at 3422.57cm -1 , while LEP / lysozyme / Ca 2+ When gel is formed, the peak shifts to 3443.97 cm -1 In LEP / TGase / Ca 2+ The characteristic peak of -OH of SSG appears at 3501.06 cm -1 ,SSG / lysozyme / Ca 2+ When gel is formed, the peak shifts to 3443.98 cm -1 , SSG / TGase / Ca 2+ When gel is formed, the peak shifts to 3419.07 cm -1 In LEP / TGase / Ca 2+ 、SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+In the gel system, the stretching vibration peak of OH moves to the low wave number direction and appears blue shift, indicating that the intermolecular hydrogen bond interaction is enhanced and the intermolecular interaction is increased, thereby improving the macroscopic properties of the composite gel system and enhancing the gel strength. 2+ The -OH groups of the ions show a red shift to higher wavenumbers, indicating a weakening of the hydrogen bonding force. This observation suggests that the hydrogen bonding may be affected by the Ca 2+ The changes in the peak intensity and peak position of the amide I band are often used to illustrate the transition of the secondary structure of proteins. The peak of the amide I region is 1700-1600 cm -1 , including α-helix (1646 -1 -1664cm -1 ), β-sheet (1615 -1 -1637 -1 and 1682 -1 -1700cm -1 ), β-turn (1664 -1 -1681cm -1 ) and random curl (1637 -1 -1645cm -1 In LEP and SSG, the absorption peaks of amide I band are located at 1644.21 cm -1 and 1648.48cm -1 , but in the four composite gels, these absorption peaks shifted to 1645.63 cm -1 (LEP / lysozyme / Ca 2+ )、1634.21cm -1 (LEP / TGase / Ca 2+ ) and 1651.34cm -1 (SSG / lysozyme / Ca 2+ ) and at 1635.64cm -1 (SSG / TGase / Ca 2+ ) in the four gel systems.

[0118] Experiment 4: X-ray diffractometer analysis

[0119] XRD analysis is an important technique for characterizing the crystalline state of a substance and studying the structure and properties of crystalline substances at the molecular level.

[0120] Sample: Take composite gel SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+ 、LEP / lysozyme / Ca 2+and LEP / TGase / Ca 2+ ; Low ester pectin LEP and Artemisia sphaerocephala seed gum polysaccharide SSG.

[0121] Test method: The freeze-dried samples were ground into powder and the crystallinity of the samples was analyzed by X-ray diffraction (XRD, D8 Discover A25, Brooker, Germany). Figure 4 shown.

[0122] See also Figure 4 , revealing the strong hydrogen bonding and crystallinity exhibited by LEP and SSG. LEP exhibits obvious crystal peaks at approximately 20° and 34°, while SSG shows a sharp crystal peak at approximately 20°. Diffraction peaks are usually observed in crystalline materials. However, LEP / lysozyme / Ca 2+ and SSG / lysozyme / Ca 2+ The diffraction peaks of the composite gel decreased, indicating that Ca 2+ The cross-linking of LEP may destroy the crystal structure of LEP, resulting in a decrease in the intensity of the diffraction peak and a transition to an amorphous state. This phenomenon is due to the fact that Ca 2+ After adding TGase, LEP / TGase / Ca 2+ There is a significantly weakened diffraction peak at about 28°, while SSG / TGase / Ca 2+ The gel showed almost negligible diffraction peaks. TGase can form intermolecular and intramolecular covalent cross-links between protein molecules to form amorphous aggregates, which is due to the multifunctional role of TGsae as a protein and a cross-linker, thereby promoting the formation of a more compact core-shell structure.

[0123] Experiment 5: Differential Scanning Calorimetry Analysis

[0124] Sample: Take composite gel SSG / lysozyme / Ca 2+ 、SSG / TGase / Ca 2+ 、LEP / lysozyme / Ca 2+ and LEP / TGase / Ca 2+ ; Low ester pectin LEP and Artemisia sphaerocephala seed gum polysaccharide SSG.

[0125] Test method: Take 2 mg of the above vacuum-dried hydrogel sample and place it in a crucible under nitrogen protection for scanning. At the same time, scan an empty aluminum crucible as a blank control. Use DSC for analysis, slowly increase the temperature at a rate of 15°C / min, and the temperature range is 25°C-150°C. The DCS analysis results are as follows: Figure 5 shown.

[0126] See also Figure 5 It can be seen that the heat resistance temperature of low ester pectin LEP is 75°C, the heat resistance temperature of Artemisia sphaerocephala seed gum polysaccharide SSG is 79°C, and the heat resistance temperature of the composite gel prepared in Examples 1 to 4 of the present invention is 102°C to 112.5°C. This shows that the composite gel prepared in the present invention can enhance the heat tolerance of the hydrogel material.

[0127] Experiment 6: Encapsulation efficiency analysis

[0128] Sample: Take the colon-targeted composite gel loaded with succinic acid prepared in Examples 1 to 4.

[0129] Then, the SA-loaded composite gel was washed 5 times with 5 mL of deionized water, and the washing liquid was collected, mixed, and 1 mL was drawn to detect SA by LC / MS. The determination was repeated three times, and the average value was calculated. The results are shown in Table 2.

[0130] Table 2 Succinic acid encapsulation efficiency of four protein / polysaccharide composite gels

[0131] <![CDATA[Ca 2+ Content]]> Material Encapsulation efficiency 8％ <![CDATA[LEP / lysozyme / Ca 2+ ]]> 87.75±0.41％ 8％ <![CDATA[SSG / lysozyme / Ca 2+ ]]> 89.05±0.27％ 8％ <![CDATA[LEP / TGase / Ca 2+ ]]> 92.07±0.30％ 8％ <![CDATA[SSG / TGase / Ca 2+ ]]> 98.92±0.10％

[0132] As can be seen from Table 2, the content of lysozyme and Ca 2+ The encapsulation efficiency of the gel was greater than 80%, among which SSG / lysozyme / Ca 2+ The encapsulation efficiency of the gel was slightly higher than that of LEP / lysozyme / Ca 2+ Composite gel, indicating that SSG is superior to LEP in encapsulation; containing TGase and Ca 2+ The encapsulation efficiency of the composite gel can exceed 90%, exceeding that of the lysozyme / Ca 2+ SSG / TGase / Ca 2+ The encapsulation efficiency of the gel is higher than that of LEP / TGase / Ca 2+ The gel showed that the gel with the addition of TGase showed better encapsulation effect. At the same time, the aforementioned SEM and FT-IR analysis also confirmed that cross-linking with TGase can form a denser and more stable structure, thereby enhancing the thermal stability of the composite gel. These findings indicate that the addition of TGase promotes the interaction force and effectively encapsulates succinic acid (SA).

[0133] Test 7: In vitro simulated release

[0134] In vitro release studies were conducted on the four composite gels prepared in this example to verify their applicability as colon-targeted SA release systems.

[0135] Sample: Take the colon-targeted composite gel loaded with succinic acid prepared in Examples 1 to 4.

[0136] 3 g of each of the four groups of gel samples were taken and released in 20 mL of simulated gastric fluid (SGF, pH = 1.2) for 2 h, then the gel was taken out and placed in simulated small intestinal solution (SIF, pH 6.8) for 2 h, and then the gel was taken out and placed in simulated colon solution (SCF, pH 7.4) for 2 h. The three stages of simulated release were all carried out at 37.1 ° C and 100 r / min.

[0137] The process was carried out in a shaker. 2 mL of release solution was drawn at 0 min, 15 min, 20 min, 60 min, and 120 min in the three release stages. At the same time, 2 mL of new simulated release solution was added to detect the SA concentration in the release solution sample.

[0138] The release rate curves of succinic acid (SA) of four composite gels loaded with succinic acid in simulated gastric fluid (SGF), simulated small intestinal fluid (SIF) and simulated colonic fluid (SCF) are shown in Figure 2. Figure 6 As shown, where: Figure 6 A is the SA release rate in SGF(a), Figure 6 B is the SA release rate in SIF (b), Figure 6 C is the SA release rate in SCF (c).

[0139] See also Figure 6 , among which SSG / TGase / Ca 2+ The 2h release rate of SA in the composite gel in SCF was 33.36±0.24%. The results showed that the cross-linking of SSG and TGase during the preparation process formed a gel network structure, which effectively protected SA and promoted its controlled release in SCF. The composite gel prepared by lysozyme began to release in SGF within the first 15min, and the release rate reached a peak of 55.99±1.21% after 2h. In the composite gel formed by TGase, LEP / TGase / Ca 2+ The composite gel released significantly after 30 min, and the release rate reached 38.57±4.54% in 2 h. 2+ Among the four composite gels, the 2h release rate in SGF was the lowest, which was 22.79±0.16%. This is because under the acidic conditions of the stomach, the -NH 3+Due to the electrostatic repulsion caused by the positive charge below pH, the protein can be hydrolyzed by pepsin proteolysis under gastric conditions, and the addition of TGase can form covalent interactions. Therefore, a tighter network structure is produced under gastric conditions, delaying the proteolysis of proteins. In SIF, the four composite gels released a large amount of SA after 30 min, because the pancreatic enzymes in SIF caused the gel to rupture and the SA therein was released. After soaking in SGF and SIF for 2 h, the four composite gels had different release rates in SCF. It is worth noting that the composite gel formed by SSG and TGase showed a higher release rate (33.36±0.25%), which was attributed to the enzymatic action of SCF, which promoted the hydrolysis of pectin and subsequent SA release.

[0140] The results of encapsulation efficiency and in vitro simulated release showed that SSG / TGase / Ca 2+ The combination of composite gels had the highest colon-targeted release efficiency and the largest encapsulation rate among the four composite gels in simulated colon fluid. This is because the intermolecular hydrogen bonding interactions of the combination of SSG and TGase were enhanced, and the intermolecular interactions increased, thereby improving the macroscopic properties of the composite gel system, enhancing the gel strength, and achieving the colon-targeted delivery effect of SA.

[0141] Experiment 8: Animal model experiment

[0142] The SSG / TGase composite gel with the highest colon release rate was selected for animal testing.

[0143] In this study, a food-safe colon-targeted gel carrier (Oral Colon Targeted Delivery System, OCDS) was constructed using proteins and polysaccharides as raw materials, aiming to control the colon-targeted release of SA and to functionally evaluate the glucose-control effect in whole-body mouse experiments.

[0144] Based on the establishment of high-sugar and high-fat diet model mice, T2DM model mice (high-fat and high-sugar mice) were induced by injection of streptozotocin (STZ). OCDS-SA intervention was performed for 8 weeks to explore the effect of colon-targeted SA supplementation on the activation ability of IGN and glucose metabolism-related indicators of T2DM mice.

[0145] The following model groups were set up: NC group (blank control group), T2DM group (high-fat and high-sugar diet group), T2DM+400 mg / kg OCDS-SA group, and T2DM+metformin group (MET group).

[0146] The MET group was given 100 mg / kg of metformin (Met) intervention on the basis of high-fat and high-sugar diet, and the OCDS-SA group was given 400 mg / kg of SA intervention on the basis of high-fat and high-sugar diet.

[0147] The above four model groups were intervened for 8 weeks and the following analysis and testing were carried out. The results are as follows.

[0148] 8.1. Changes in body weight, liver and kidney weights

[0149] During the 8-week intervention, the average body weight of mice in each group was measured and calculated weekly. Figure 7 After 8 weeks of intervention, the mice were treated with conventional methods, and the average weight of the liver and kidney of each group of mice was calculated. Figure 8 As shown, (a) is the comparison of the average liver weight of each group; (b) is the comparison of the average kidney weight of each group.

[0150] See also Figure 7 and Figure 8 The results showed that the intervention of OCDS-SA can effectively reverse the sudden weight loss of T2DM mice, and significantly inhibit the abnormal increase in liver and kidney weight of T2DM mice, avoid liver damage, and make them tend to normal.

[0151] 8.2 Serum Indicators

[0152] After 8 weeks of intervention, blood was collected from mice using conventional methods, and serum indicators of each group were calculated. The results are as follows: Fig. 9 As shown; (a) is the comparison of the average high-density lipoprotein cholesterol values ​​of each group; (b) is the comparison of the average low-density lipoprotein cholesterol values ​​of each group; (c) is the comparison of the average total cholesterol values ​​of each group; (b) is the comparison of the average triglyceride values ​​of each group.

[0153] See also Fig. 9 OCDS-SA can significantly reduce the levels of serum low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and triglyceride (TG) in the basic indicators of T2DM mice (p < 0.05), and significantly increase the content of serum high-density lipoprotein cholesterol (HDL-C) (p < 0.05).

[0154] 8.3 Blood Glucose Metabolism Indicators

[0155] After 8 weeks of intervention, blood was collected from mice using conventional methods, and the blood glucose metabolism indexes of each group were calculated. The results are as follows: Fig.10 As shown; among which: Fig.10 A is the comparison of the mean blood glucose values ​​(ITT) of each group; Fig.10 B is the comparison of the mean values ​​of glucose tolerance (OGTT) in each group; Fig.10 C is the comparison of the average results of insulin sensitivity in each group.

[0156] from Fig.10 It can be seen that in terms of regulating blood sugar metabolism, OCDS-SA intervention reduced blood sugar levels, glucose tolerance and insulin sensitivity in T2DM mice, activated IGN in type 2 diabetes model (T2DM) mice, and regulated their blood sugar disorders.

[0157] See also Fig.21 , significantly increased the mRNA expression of gluconeogenesis-related genes G6Pase, PEPCK, SREBP-1c and Fas in the colon tissue of T2DM mice (p < 0.05), among which the mRNA expression of G6Pase, PEPCK and Fas genes was significantly increased compared with the NC group.

[0158] 8.4. Intestinal flora

[0159] See also Figure 11 to Figure 14 The test examined the effect of the gel on various parameters of the intestinal flora.

[0160] Ginseng Figure 15-16 It was found that T2DM model mice had intestinal flora disorder, and OCDS-SA treatment could significantly improve the Shannon index and Chao index of T2DM mice (p < 0.05), and increase the diversity of intestinal flora in T2DM mice.

[0161] Fig.17 The results of short-chain fatty acids are mainly used to test the performance of the gel. The SCFAs content in the small intestine, colon, and cecum of mice was detected by high performance liquid chromatography-mass spectrometry. The SCFAs content has a great influence on intestinal flora and glucose and lipid metabolism.

[0162] 8.5. Succinate delivery function

[0163] The content of succinic acid in the intestine was detected by high performance liquid chromatography-mass spectrometry. Figure 18 to Figure 20 It can be seen that the composite gel provided by the present invention can increase the content of succinate SA in the intestine by targeted delivery of succinate SA.

[0164] Through the above experiments, the composite gel prepared in this example can improve the targeted release rate of succinic acid in the colon, can enhance the mRNA expression of genes, and can also improve the Shannon index and Chao index of type 2 diabetes model mice, and can be used in the preparation of drugs for treating colitis.

[0165] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a colon-targeted composite gel, characterized in that: The following steps are involved: S1. Dissolve the polysaccharide and the protein in equal volumes of water to obtain a polysaccharide solution and a protein solution respectively; the mass ratio of the polysaccharide to the protein is (1.9-2.1):1; S2. Mix the protein solution and the polysaccharide solution, then add succinic acid and calcium chloride, add 0.05wt%-0.1wt% succinic acid and 6wt%-8wt% calcium chloride to every 10ml of protein solution; finally, incubate at 4±0.5°C overnight to obtain a colon-targeted composite gel.

2. The method for preparing the colon targeting composite gel according to claim 1, characterized in that: The polysaccharide is artemisia seed gum polysaccharide, low-ester pectin, chitosan or inulin.

3. The method for preparing the colon targeting composite gel according to claim 1, characterized in that: The protein is lysozyme, glutaminase or whey protein isolate.

4. The colon-targeted composite gel prepared by the preparation method of the colon-targeted composite gel according to claim 1.

5. The colon targeting composite gel according to claim 4, characterized in that: The encapsulation rate of the colon-targeted composite gel for succinic acid reaches 87.75% to 98.92%.

6. Use of the colon-targeted composite gel according to claim 4 in preparing a drug for treating colitis.

7. Use of the colon-targeted composite gel according to claim 4 in preparing a drug for treating colitis by improving the targeted release rate of succinic acid.

8. Use of the colon-targeted composite gel according to claim 4 in preparing a drug for treating colitis by enhancing gene mRNA expression.

9. The use according to claim 8, characterized in that: The genes are G6Pase, PEPCK, SREBP-1c and / or Fas.

10. Use of the colon-targeted composite gel according to claim 4 in preparing a drug for treating colitis by improving the Shannon index and Chao index of type 2 diabetes model mice.

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