A colon-targeted composite gel and its preparation method and application

Abstract

The present invention belongs to the technical field of composite gels and their applications, and relates to a colon-targeted composite gel, its preparation method, and application. The preparation method of the colon-targeted composite gel comprises the following steps: S1, dissolving a polysaccharide and a protein in equal volumes of water, respectively, to obtain a polysaccharide solution and a protein solution; S2, mixing the protein solution and the polysaccharide solution, adding succinic acid and calcium chloride, and finally incubating at 4°C overnight to obtain a colon-targeted composite gel. The present invention utilizes polysaccharide, protein, succinic acid, and calcium chloride to form a colon-targeted composite gel, which has a dense and stable structure, greatly improving the encapsulation efficiency of succinic acid, facilitating the targeted release rate of succinic acid in the colon, and achieving precise targeted delivery of succinic acid in the colon.

Description

Technical Field

[0001] The invention belongs to the technical field of composite gels and their applications, 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 changes in people's dietary habits, obesity and impaired glucose metabolism have become increasingly serious problems worldwide in recent years, with the number of patients showing an upward trend year by year. Currently, the treatment of diabetes relies primarily on oral hypoglycemic medications and insulin injections. In terms of preventive measures, other than dietary adjustments, there is currently a lack of more effective means to prevent the onset of diabetes.

[0003] Recent studies have revealed that the intestine has the ability to produce gluconeogenesis (IGN) and can negatively feedback regulate blood glucose disorders through the gut-brain-liver axis, achieving the effect of controlling appetite and regulating blood glucose levels. Therefore, intestinal gluconeogenesis (IGN) is an important pathway for the body to regulate glucose metabolism. In the IGN pathway, succinic acid (SA) is a key substance that activates and participates in IGN. 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 gastrointestinal burden, and cause symptoms such as abdominal distension. If small molecule carboxylic acid SA is ingested orally, SA is easily absorbed by the small intestine into the systemic circulation, potentially inducing adverse reactions such as tissue and organ damage and inflammation, making it difficult to reach the colon and activate IGN. Therefore, it is crucial and important to precisely deliver SA to the colon to activate IGN and regulate blood glucose homeostasis.

[0004] There is an existing method for delivering a target object to the colon. Referring to the Chinese patent document CN118109536A, a method for preparing nano-resistant pure amylose barley starch is disclosed, which includes enzymatically hydrolyzing pure amylose barley starch with a buffer solution containing barley malt 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 the 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 the aqueous solution containing the nano-resistant pure amylose barley starch stable complex as the aqueous phase and the oil phase containing the fat-soluble drug as the 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 the targeted colon release of the emulsion.

[0005] However, the existing methods for encapsulating succinic acid have a low encapsulation rate due to poor stability, 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 efficiency and the inability to achieve precise targeted delivery of succinic acid in the colon, the present invention provides a colon-targeted composite gel and a preparation method and application thereof.

[0007] The present invention uses polysaccharides, proteins, 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 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, adding 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] It is further defined that 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 efficiency of the colon-targeted composite gel for succinic acid reaches 87.75% to 98.92%.

[0016] The colon-targeted composite gel is used in the preparation of medicines for treating colitis.

[0017] The colon-targeted composite gel is used in preparing medicine 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 increasing gene mRNA expression.

[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 uses polysaccharides, proteins 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 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 reaches up to 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 glucose 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 them, the mRNA expression of G6Pase, PEPCK and Fas genes was significantly increased compared with the NC group. At the same time, it also activated the IGN of T2DM mice, and had the ability to regulate blood glucose.

[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 the intestinal flora of 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 calcium ion-containing polysaccharide / protein composite gels;

[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 are shown;

[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 are shown;

[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 Comparison of weight results among groups;

[0034] Figure 8 Comparison of liver and kidney results among the groups;

[0035] Figure 9 The comparison results of serum indicators in each group are shown;

[0036] Figure 10 Comparison of blood sample index analysis results for each group;

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

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

[0039] Figure 13 is the Kruskal-Wallis test plot of the intestinal flora of each group;

[0040] Figure 14 Columnar analysis of intestinal flora in each group;

[0041] Figure 15 Results for the mouse intestinal flora;

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

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

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

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

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

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

[0048] The technical solutions protected by the present invention are now described in detail with reference to the accompanying drawings and embodiments. However, it is apparent that the described embodiments are only a portion of the embodiments of this application, and not all of them. Based on the described embodiments of this application, all other embodiments derived by persons of ordinary skill in the art without requiring creative effort are also within the scope of protection of this 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 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, adding 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 above-mentioned 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, it can improve the targeted release rate of succinic acid in the colon, enhance the mRNA expression of genes, and improve the Shannon index and Chao index of type 2 diabetes model mice.

[0055] The technical solutions and performances protected by 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 apples and purchased from Shandong Qilu Biotechnology Co., Ltd. Artemisia ordosica seed polysaccharide (SSG) was purchased from Hubei Gumu Biotechnology Co., Ltd. Calcium chloride (CaCl2), simulated gastric fluid (containing pepsin), simulated small intestinal fluid (containing trypsin and phosphate), simulated colonic fluid, transglutaminase (TGase), lysozyme, and succinic acid (SA) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd. All conventional chemical reagents were of analytical grade.

[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 automated 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 Ultramicrospectrophotometer NanoDrop One Thermo Fisher Scientific Stable blood glucose meter Stable Sinocare Biosensing Co., Ltd. PCR instrument CFX Connect Optics Module Bio-rad microplate reader HM-SY96S Hengmei Fourier transform infrared spectroscopy INVENNIO S BRUKER DSC microscope system DSC1 Professional Mettler Toledo X-ray diffractometer D8 DISCOVER A25 BRUKER Field emission scanning electron microscopy Nava NanoSEM 450 Thermo Fisher Scientific

[0063] Example 1

[0064] The preparation method of the colon-targeted 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 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 the solution is completely dissolved to obtain a protein solution.

[0068] S2. The protein solution and polysaccharide solution were mixed, and succinate SA and calcium chloride CaCl2 were added. 0.05 g succinate 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, which was recorded as SA-loaded SSG / lysozyme / CaCl2. 2+ Composite gel.

[0069] Example 2

[0070] The preparation method of the colon-targeted 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 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 completely dissolved to obtain a protein solution.

[0074] S2. The protein solution and polysaccharide solution were mixed, and succinate SA and calcium chloride CaCl2 were added. 0.05 g succinate 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, which was recorded as SA-loaded SSG / TGase / CaCl2. 2+ Composite gel.

[0075] Example 3

[0076] The preparation method of the colon-targeted 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 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 the solution is completely dissolved to obtain a protein solution.

[0080] S2. The protein solution and the polysaccharide solution were mixed, and succinate SA and calcium chloride CaCl2 were added. 0.05 g succinate 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, which was recorded as SA-loaded LEP / lysozyme / CaCl2. 2+ Composite gel.

[0081] Example 4

[0082] The preparation method of the colon-targeted 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 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 completely dissolved to obtain a protein solution.

[0086] S2. The protein solution and the polysaccharide solution were mixed, and succinate SA and calcium chloride CaCl2 were added. 0.05 g succinate 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, which was recorded as SA-loaded LEP / TGase / CaCl2. 2+ Composite gel.

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

[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 in the experiment for comparison.

[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. 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 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. Then, 0.8 g of CaCl2 was added and stirred until it was 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 Artemisia seed gum polysaccharide SSG or low-ester pectin LEP, and the protein is lysozyme lysozyme or transglutaminase TGase.

[0096] SSG / lysozyme / Ca 2+ Composite gel, SSG / TGase / Ca 2+ Composite gel, LEP / lysozyme / Ca 2+ 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. 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 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 heating, 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 preparation, the polysaccharide is Artemisia seed gum polysaccharide SSG or low-ester pectin LEP, and the protein is lysozyme. SSG / lysozyme composite gel and LEP / lysozyme / composite gel are prepared respectively according to the above method.

[0104] The physical and chemical properties tests are used to compare and analyze the properties of the composite gel materials prepared in this embodiment and the comparative example.

[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 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). The measurement was repeated three times. Figure 1 Above: SSG / lysozyme, SSG / TGase, LEP / lysozyme, and LEP / TGase refer to composite gels containing calcium ions.

[0108] See also Figure 1The average particle size of the four cross-linked composite gels was significantly larger than that of LEP and SSG (p < 0.05). This is partly because the composition of the protein-polysaccharide composite gels undergoes a transition from two single substances to a mixture. Furthermore, the protein is preheated (85°C) during the preparation of the composite gels, which unfolds the protein molecules, fully exposing their hydrophobic groups. Under weak interactions such as hydrophobic bonds and disulfide bonds, the proteins swell and aggregate to form protein aggregates, which then cross-link with the polysaccharides. This entire process involves changes in the protein conformation within the composite particles, affecting their cross-linking with LEP and SSG and forming complexes larger than those of the single proteins or polysaccharides.

[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: The microstructure of each freeze-dried gel sample was observed using a field emission scanning electron microscope (FEI, Nava Nano SEM 450). The freeze-dried gel sample was fixed on a conductive double-sided tape, sprayed with 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, after adding protein cross-linked gel, the lamellar structure disappears and is replaced by a smoother and denser structure. This 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 / Ca2+ and LEP / TGase / Ca 2+ ; Low ester pectin LEP and Artemisia seed gum polysaccharide SSG.

[0115] Testing Method: Fourier transform infrared spectroscopy (FTIR, Invennio S, Brooker, Germany) was used to determine molecular interactions within proteoglycan gels. FT-IR is a commonly used instrument for analyzing interactions within substances. By measuring the sample's absorption of infrared radiation at various wavelengths, it clearly reveals information about the sample's functional groups and interactions within the substance. It offers the advantages of simplicity, rapidity, non-destructiveness, high efficiency, and environmental friendliness.

[0116] Specifically, the KBr tablet method was used. 3 mg of freeze-dried gel sample was accurately weighed and mixed with 197 mg of KBr, ground into a fine powder, and pressed 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 as follows Figure 3 shown.

[0117] See also Figure 3 , 3100 -1 -3500cm -1 The band is the stretching vibration of hydroxyl groups, and the characteristic peak of -OH in LEP appears at 3422.57 cm -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 toward 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 OH groups 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 transformation of the secondary structure of proteins. The peak of the amide I region is 1700-1600 cm -1 , including α-helices (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+ ) position. This indicates that electrostatic interactions exist in all four gel systems.

[0118] Experiment 4: X-ray diffractometer analysis

[0119] XRD analysis is an important technique for characterizing the crystalline state of substances 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 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 distinct 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, resulting in a decrease in the diffraction peak intensity and a transition to an amorphous state. This phenomenon is due to the fact that Ca 2+ The network-like eggshell structure formed after cross-linking. After adding TGase, LEP / TGase / Ca 2+ It showed 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 crosslinks between protein molecules to form amorphous aggregates. This is due to the multifunctional role of TGsae as a protein and a crosslinker, 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 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 to analyze, slowly increase the temperature at a rate of 15℃ / min, and the temperature range is 25℃-150℃. The DCS analysis results are as follows: Figure 5 shown.

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

[0127] Experiment 6: Encapsulation Efficiency Analysis

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

[0129] The SA-loaded composite gel was then rinsed five times with 5 mL of deionized water. The washes were collected, mixed, and 1 mL was aspirated for SA analysis using LC / MS. The assay was repeated three times, and the average values ​​were 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 TGase added showed better encapsulation effect. Furthermore, the aforementioned SEM and FT-IR analyses 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 interaction forces 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: The colon-targeted composite gel loaded with succinic acid prepared in Examples 1 to 4 was taken.

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

[0137] The release 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. 2 mL of new simulated release solution was added at the same time 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 4. 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 2-h 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 15 minutes, and the release rate reached a peak of 55.99±1.21% after 2 hours. 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 of 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 under gastric conditions, and the addition of TGase can form a covalent interaction. Therefore, a tighter network structure is produced under gastric conditions, delaying the proteolysis of the protein. In SIF, the four composite gels released a large amount of SA after 30 minutes, 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 hours, 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 colonic fluid. This is because the intermolecular hydrogen bond interaction of the combination of SSG and TGase was enhanced, and the intermolecular interaction 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 its glucose-control effect in whole-body mouse experiments.

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

[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 performed, and 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 OCDS-SA intervention 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 return 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: Figure 9 As shown; (a) is the comparison of the average high-density lipoprotein cholesterol of each group; (b) is the comparison of the average low-density lipoprotein cholesterol of each group; (c) is the comparison of the average total cholesterol of each group; (b) is the comparison of the average triglyceride of each group.

[0153] See also Figure 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 high-density lipoprotein cholesterol (HDL-C) in the serum (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 indicators of each group were calculated. The results are as follows: Figure 10 shown; wherein: Figure 10 A is the comparison of the mean blood glucose values ​​(ITT) of each group; Figure 10 B is the comparison of the average values ​​of glucose tolerance (OGTT) in each group; Figure 10 C is the comparison of the average insulin sensitivity results of each group.

[0156] from Figure 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 the IGN of type 2 diabetes (T2DM) mice, and regulated their blood sugar disorders.

[0157] See also Figure 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 Microflora

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

[0160] Ginseng Figures 15 and 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), increasing the diversity of intestinal flora in T2DM mice.

[0161] Figure 17 The results of short-chain fatty acids were mainly used to test the performance of the gel. The content of SCFAs in the small intestine, colon, and cecum of mice was detected by high-performance liquid chromatography-mass spectrometry. The SCFA content has a significant impact on intestinal flora 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. Figures 18 to 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, enhance gene mRNA expression, and 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 polysaccharide and protein in equal volumes of water to obtain polysaccharide solution and protein solution respectively; the mass ratio of polysaccharide to protein is (1.9-2.1):1; S2. Mix the protein solution and the polysaccharide solution, then add succinic acid and calcium chloride, adding 0.05 wt%-0.1 wt% succinic acid and 6 wt%-8 wt% calcium chloride per 10 ml of the protein solution; finally, incubate at 4±0.5° C. overnight to obtain a colon-targeted composite gel; The polysaccharide is Artemisia ordosica seed gum polysaccharide or low-ester pectin; and the protein is lysozyme or transglutaminase.

2. The colon-targeted composite gel prepared by the method for preparing the colon-targeted composite gel according to claim 1.

3. The colon-targeting composite gel according to claim 2, characterized in that: The encapsulation efficiency of the colon-targeted composite gel for succinic acid reaches 87.75% to 98.92%.

4. Use of the colon-targeted composite gel according to claim 2 in preparing a drug for treating type 2 diabetes.

5. Use of the colon-targeted composite gel according to claim 2 in preparing a drug for treating type 2 diabetes by increasing the targeted release rate of succinic acid.

6. Use of the colon-targeted composite gel according to claim 2 in preparing a drug for treating type 2 diabetes by enhancing gene mRNA expression.

7. The use according to claim 6, characterized in that The genes are G6Pase, PEPCK, SREBP-1c and / or Fas.

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

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