阅读说明：本技术 制备抵抗α-葡萄糖苷酶水解的交联右旋糖苷凝胶的方法 (Method for preparing cross-linked dextran gel resistant to hydrolysis by alpha-glucosidase ) 是由 辛瑜 于 2019-10-14 设计创作，主要内容包括：本发明提供一种制备抵抗α-葡萄糖苷酶水解的交联右旋糖苷凝胶的方法,以低分子量右旋糖苷为起始骨架,用含有环氧基团的活化剂对其进行活化,使其带有多个环氧基团,活化的低分子量右旋糖苷；所述含有环氧基的活化剂为1,4-丁二醇二缩水甘油醚、乙二醇二缩水甘油醚或环氧氯丙烷中的一种；利用活化的低分子量右旋糖苷作为生物交联中间体与高分子量右旋糖苷混合并进行交联反应形成水凝胶,并均质形成均一的凝胶颗粒；加入N-乙酰-D-氨基葡萄糖,D-氨基葡萄糖,以及纤维二糖功能单体。本方法所制备的凝胶,在体外交联右旋糖苷酶解的实验中,α-葡萄糖苷酶的催化效率被降低50％以上。(The invention provides a method for preparing cross-linked dextran gel resisting alpha-glucosidase hydrolysis, which takes low molecular weight dextran as an initial skeleton, and activates the dextran gel by an activating agent containing epoxy groups to ensure that the dextran gel has a plurality of epoxy groups and is activated; the activator containing the epoxy group is one of 1, 4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether or epichlorohydrin; mixing activated low molecular weight dextran serving as a biological crosslinking intermediate with high molecular weight dextran, carrying out crosslinking reaction to form hydrogel, and homogenizing to form uniform gel particles; adding N-acetyl-D-glucosamine, D-glucosamine and cellobiose functional monomer. The gel prepared by the method has the advantage that the catalytic efficiency of alpha-glucosidase is reduced by more than 50% in an in-vitro cross-linked dextran enzymolysis experiment.)

1. A method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase comprising:

step 1) activating low molecular weight dextran with an activating agent containing epoxy groups by using the low molecular weight dextran as an initial skeleton to enable the dextran to have a plurality of epoxy groups, so as to obtain activated low molecular weight dextran; the activator containing the epoxy group is one of 1, 4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether or epichlorohydrin;

step 2) mixing activated low molecular weight dextran serving as a biological crosslinking intermediate with high molecular weight dextran, carrying out crosslinking reaction to form hydrogel, and homogenizing to form uniform gel particles;

the molecular weight of the low molecular weight dextran is 10000-20000Da, and the molecular weight of the high molecular weight dextran is 700000-1000000 Da.

Step 3), adding N-acetyl-D-glucosamine, D-glucosamine and cellobiose functional monomers, wherein the chemical formulas are respectively as follows:

2. the method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

in the step 1), the molar ratio of the low molecular weight dextran functional monomer to the epoxy group activator is 1:4-1:10, the activation reaction time is 2-5 hours, the activation reaction temperature is 30-50 ℃, and 0.1-0.4M NaOH is used as a catalyst in the activation reaction.

3. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

in the step 2), the molar ratio of epoxy group contained in the epoxidized low molecular weight dextran biological crosslinking intermediate to the high molecular weight dextran functional monomer is 1:30-1:50, the crosslinking reaction time is 2-5 hours, the crosslinking reaction temperature is 30-50 ℃, and 0.1-0.4M NaOH is used as a catalyst in the crosslinking reaction.

4. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

after obtaining the epoxidized low molecular weight dextran biological crosslinking intermediate, purifying and removing the residual activating agent containing epoxy groups by using 4-8 times of cold ethanol precipitation and washing steps.

5. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

the final gel particle product was washed 4-8 times with normal saline.

6. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

in step 1), the concentration of the low molecular weight dextran is 20-30 wv%.

7. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

in step 2), the concentration of high molecular weight dextran was 30 wv%.

8. The method of preparing a cross-linked dextran gel resistant to hydrolysis by α -glucosidase as claimed in claim 1 wherein:

wherein the working concentration of the functional monomer is 0.2-0.6 mmol/L.

Technical Field

The invention relates to a method for preparing cross-linked dextran gel resisting alpha-glucosidase hydrolysis, and belongs to the field of materials.

Background

Dextran is a kind of polymeric polysaccharide which takes alpha-glycosidic bond as link and glucose as basic functional monomer, and can be used as plasma dilator because it has no antigenicity, can have stable structure, and can resist high-temperature and high-pressure sterilization. The dextran hydrogel has good water retention property and high stability, and has great application potential in the injection filling field of the plastic and beauty industry. Dextran has been shown to effectively enhance the immune system of the body, resist diseases caused by microorganisms, and have attracted attention for applications in the fields of tumors, infectious diseases, wound treatment and the like. In addition, dextran has effects of scavenging free radicals, resisting radiation, dissolving cholesterol, preventing hyperlipidemia, and resisting infection caused by filtering virus, fungi, bacteria, etc. Therefore, the method is widely applied to the industries of medicines, foods, cosmetics and the like.

In the human body, alpha-Glucosidase (EC 3.2.1.20) is an enzyme capable of hydrolyzing alpha-glucosidic bonds, and can specifically hydrolyze alpha-1, 6/1,3/1 and 4 glucosidic bonds in dextran molecules, thereby degrading the dextran molecules. Currently, injection solutions thereof have been used for the treatment of patients with α -glucosidase deficiency.

Because the dextran has the characteristics of stable structure, no antigenicity and the like, a mild and efficient crosslinking method can be developed to prepare the crosslinked dextran gel for filling injection in the cosmetic industry. In addition, the human body expresses the alpha-glucosidase by itself, so that the filled cross-linked dextran gel can be hydrolyzed and gradually loses the plasticity effect. Therefore, in order to improve the overall retention time and the molding effect of the cross-linked dextran gel in the body, it is necessary to consider the specific method for inhibiting the enzymatic hydrolysis efficiency of human α -glucosidase. At present, the maintenance time of polysaccharide-permeable hydrogel is improved only by using a mode of improving the chemical crosslinking degree, but the subsequent adverse reaction can be caused by the increase of the content of the chemical crosslinking agent.

Disclosure of Invention

The object of the present invention is to provide a method for preparing a cross-linked dextran gel resistant to hydrolysis by alpha-glucosidase in order to solve the above problems.

The invention adopts the following technical scheme:

a method for preparing cross-linked dextran gel resisting alpha-glucosidase hydrolysis comprises the steps of taking low molecular weight dextran (10000-20000Da) as an initial framework, activating the dextran gel by an activating agent containing epoxy groups to enable the dextran gel to have a plurality of epoxy groups, and obtaining a biological intermediate, namely the activated low molecular weight dextran; the activator containing the epoxy group is one of 1, 4-butanediol diglycidyl ether (BDDE), Ethylene Glycol Diglycidyl Ether (EGDE) or epichlorohydrin; mixing activated low-molecular-weight dextran serving as a biological crosslinking intermediate with high-molecular-weight dextran (700000-1000000Da) to perform a crosslinking reaction to form hydrogel, and homogenizing to form uniform gel particles; the N-acetyl-D-glucosamine, cellobiose and other functional monomers are utilized to inhibit the catalytic activity of alpha-glucosidase and prolong the stabilization time of the cross-linked dextran gel.

The method mainly comprises the following steps:

1) activating dextran with low molecular weight (10000-20000Da) as initial skeleton with activator containing epoxy group to make it have multiple epoxy groups, and obtaining a biological intermediate, i.e. activated dextran with low molecular weight; the activator containing the epoxy group is one of 1, 4-butanediol diglycidyl ether (BDDE), Ethylene Glycol Diglycidyl Ether (EGDE) or epichlorohydrin;

2) mixing activated low-molecular-weight dextran serving as a biological crosslinking intermediate with high-molecular-weight dextran (700000-1000000Da) to perform a crosslinking reaction to form hydrogel, and homogenizing to form uniform gel particles;

3) the N-acetyl-D-glucosamine, cellobiose and other functional monomers are utilized to inhibit the catalytic activity of alpha-glucosidase and prolong the stabilization time of the cross-linked dextran gel. Preferably, in the step 1), the molar ratio of the low molecular weight dextran functional monomer to the epoxy group activator is 1:4-1:10, the activation reaction time is 2-5 hours, the activation reaction temperature is 30-50 ℃, and 0.1-0.4M NaOH is used as a catalyst in the activation reaction.

Preferably, in the step 1), the concentration of the low molecular weight dextran is 20-30 wv%, the molar ratio of epoxy groups contained in the epoxidized low molecular weight dextran biological crosslinking intermediate to the high molecular weight dextran functional monomer is 1:30-1:50, the crosslinking reaction time is 2-5 hours, the crosslinking reaction temperature is 30-50 ℃, and 0.1-0.4M NaOH is used as a catalyst in the crosslinking reaction.

Preferably, in step 2), the concentration of high molecular weight dextran is 30 wv%.

Preferably, in the step 1), after the epoxidized low molecular weight dextran biological crosslinking intermediate is obtained, the step of washing with cold ethanol precipitation is performed for 4 to 8 times, and the residual activator containing epoxy groups is purified and removed.

Preferably, in step 2), the final gel particle product is washed 4-8 times with physiological saline.

Preferably, in step 3), the optimal inhibition concentration of the functional monomers such as N-acetyl-D-glucosamine, D-glucosamine and cellobiose on the catalytic activity of the alpha-glucosidase is 0.2-0.6 mmol/L.

Advantageous effects of the invention

The invention provides a method for preparing cross-linked dextran gel resisting alpha-glucosidase hydrolysis, and provides a group of methods for reducing alpha-glucosidase activity by using functional monomers such as N-acetyl-D-glucosamine, D-glucosamine and cellobiose, and effectively enhancing the maintenance time and plastic effect of cross-linked dextran hydrogel. By using the method, the catalytic efficiency of the alpha-glucosidase is reduced by more than 50% in an in-vitro cross-linked dextran enzymolysis experiment. Has larger application potential and embodies larger economic benefit.

Drawings

FIG. 1 is the interaction of N-acetyl-D-glucosamine with the catalytic central amino acid residue of human alpha-glucosidase;

FIG. 2 is the interaction of D-glucosamine with the catalytic central amino acid residue of human alpha-glucosidase;

FIG. 3 is the interaction of cellobiose with the catalytic central amino acid residue of human alpha-glucosidase;

FIG. 4 shows that N-acetyl-D-glucosamine at different concentrations inhibits the ability of human recombinant alpha-glucosidase to hydrolyze cross-linked dextran;

FIG. 5 shows the ability of D-glucosamine at various concentrations to inhibit the hydrolysis of cross-linked dextran by human recombinant α -glucosidase;

FIG. 6 shows that different concentrations of cellobiose inhibit the ability of human recombinant alpha-glucosidase to hydrolyze cross-linked dextran.

Detailed Description