阅读说明：本技术 一种固定化脂肪酶催化柠檬酸功能化β-环糊精及其制备方法 (Immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin and preparation method thereof ) 是由 赵启成 赵宇 王景昌 詹世平 于 2021-01-21 设计创作，主要内容包括：一种固定化脂肪酶催化柠檬酸功能化β-环糊精及其制备方法,属于载体材料领域。本发明采用β-环糊精和柠檬酸为原料,固定化脂肪酶为催化剂,通过酯化反应制备得到了含有羧基的β-环糊精,可以改善β-环糊精的活性和亲水性。本发明采用β-环糊精和柠檬酸为原料,固定化脂肪酶为催化剂,产物的活性和亲水性得到进一步的提高,其制备方法具有原料和催化剂绿色、反应温度温和和接枝率高的特点,且接枝率测试方法简便有效。(An immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin and a preparation method thereof, belonging to the field of carrier materials. The beta-cyclodextrin containing carboxyl is prepared by taking beta-cyclodextrin and citric acid as raw materials and immobilized lipase as a catalyst through esterification reaction, and the activity and the hydrophilicity of the beta-cyclodextrin can be improved. The invention adopts beta-cyclodextrin and citric acid as raw materials, immobilized lipase as a catalyst, the activity and the hydrophilicity of the product are further improved, the preparation method has the characteristics of green raw materials and catalysts, mild reaction temperature and high grafting rate, and the grafting rate test method is simple, convenient and effective.)

1. A preparation method of immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin is characterized by comprising the following steps: weighing beta-cyclodextrin, adding the beta-cyclodextrin into a three-neck flask containing 8mL of dimethyl sulfoxide, slowly adding citric acid into the flask, controlling the pH value of the solution, adding immobilized lipase, heating, reacting for 8-20 hours under stirring at the speed of 1500r/min, pouring a product into the flask after the reaction is finished, dissolving the product back in a solution of absolute ethyl alcohol/deionized water, centrifuging, and drying the product in a vacuum drying box at the temperature of 50 ℃ to obtain a solid powdery product beta-CD-COOH.

2. The preparation method of the immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin of claim 1, wherein the addition amount of the beta-cyclodextrin is 3g, and the addition amount of the dimethyl sulfoxide is 8 mL.

3. The method for preparing the immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin as claimed in claim 1, wherein the pH value of the solution before immobilization of the lipase is 6-8.

4. The preparation method of the immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin as claimed in claim 1, wherein the solution ratio of absolute ethanol/deionized water is absolute ethanol with the volume ratio as follows: deionized water 65: 35.

5. The method of claim 1, wherein the immobilized lipase catalyzes the preparation of citric acid functionalized beta-cyclodextrin, and the amount of the immobilized lipase added is 13 mg.

6. The immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin prepared by the preparation method of claim 1.

Technical Field

The invention belongs to the field of carrier materials, and particularly relates to immobilized lipase-catalyzed citric acid functionalized beta-cyclodextrin and a preparation method thereof.

Background

Beta-cyclodextrin is cyclic oligosaccharide with 7D-glucopyranoside units linked by 1, 4-glycosidic bonds, has the advantages of amphipathy, good biocompatibility, low immunogenicity, wide sources and low price, can improve the solubility and stability of drugs, strengthen the drug absorption, cover unnecessary odor and taste, control the drug release, improve the permeability of drugs through biological barriers (carriers), and is widely applied to the fields of pharmaceutical preparations, foods, spices, cosmetics, packaging, textiles and the like. However, the surface of the beta-cyclodextrin lacks carboxyl, and the beta-cyclodextrin is difficult to combine with a compound with amino to synthesize a new compound, and the activity and the hydrophilicity of the beta-cyclodextrin can be improved by adopting a grafting modification method. Many compounds can be used for graft modification, and citric acid is selected as a modifier of beta-cyclodextrin because of the advantages of natural non-toxicity, strong reaction activity and easy obtainment of raw materials; the lipase is prepared by deep fermentation of a genetically modified Aspergillus oryzae (Aspergillus oryzae) microorganism and adsorption onto macroporous acrylic resin. Novozym435 is an immobilized preparation of thermostable lipase, is a triacylglycerol hydrolase, and is particularly suitable for synthesis of ester and amine compounds. The positional specificity of Novozym435 depends on the kind of the reactant. At present, most of esterification reactions are catalyzed and synthesized by using metal catalysts, but most of the reactions can generate byproducts and corresponding pollution, and the biological enzyme catalysis method has the characteristics of high catalytic activity, mild conditions and environmental protection. Compared with the traditional catalytic reaction in water phase, the lipase catalysis in organic phase enlarges the application range of the enzyme. The invention adopts beta-cyclodextrin and citric acid as raw materials, immobilized lipase as a catalyst, the activity and the hydrophilicity of the product are further improved, and the preparation method has the characteristics of green raw materials and catalyst, mild reaction temperature and high grafting rate. The citric acid-beta-cyclodextrin grafting compound prepared by the invention can be used as a carrier material for targeted drug delivery.

Disclosure of Invention

Aiming at the defects, the invention provides the immobilized lipase catalyzed citric acid functionalized beta-cyclodextrin and the preparation method thereof, and the preparation method has the characteristics of green raw materials and catalysts, mild reaction temperature and high grafting rate.

The method for solving the technical problem comprises the following steps:

accurately weighing 3g of beta-CD, adding the beta-CD into a 25mL three-neck flask containing 8mL of dimethyl sulfoxide, slowly adding 1.739g of Citric Acid (CA) into the flask, controlling the pH value of the solution within the range of 6-8, adding 4-13mg of immobilized lipase, heating to a certain temperature, stirring at the speed of 1500r/min, reacting for a certain time, pouring a product into a beaker after the reaction is finished, performing back dissolution by using a solution with the volume ratio of anhydrous ethanol/deionized water of 65/35, centrifuging, and drying the product in a vacuum drying oven at the temperature of 50 ℃ to obtain a solid powdery product of beta-CD-COOH.

The principle is as follows: the invention relates to a method for catalyzing citric acid functionalized beta-cyclodextrin by immobilized lipase, which adopts beta-cyclodextrin (beta-CD) and Citric Acid (CA) as raw materials and immobilized lipase as a catalyst to prepare beta-cyclodextrin (beta-CD-COOH) containing carboxyl through esterification reaction, and can improve the activity and the hydrophilicity of the beta-cyclodextrin. The influence of the reaction temperature, the reaction time and the catalyst amount on the grafting rate is explored, and the grafting rate of the product is analyzed by adopting an infrared spectroscopy; synthesizing under different conditions to obtain a series of citric acid-beta-cyclodextrin grafting compounds, and obtaining the optimal synthesis conditions. The product is subjected to structure and performance analysis by adopting an infrared spectrum, nuclear magnetism and contact angle instrument.

The method for measuring the grafting ratio of the beta-cyclodextrin and the citric acid comprises the following steps: and (3) according to the Beer-Lambert law, quantitatively analyzing the grafting rate of the product by adopting a quantitative analysis method of infrared spectrum. During measurement, firstly, a mixture of beta-cyclodextrin and citric acid mixed in different proportions is prepared, the relative content of the beta-cyclodextrin and the citric acid is determined by measuring the characteristic absorption peak area ratio of the beta-cyclodextrin and the citric acid in an infrared spectrogram of the mixture, a series of mixtures in different proportions are plotted with a series of obtained corresponding characteristic peak area ratios, the standard curve of the relative content of the beta-cyclodextrin and the citric acid is obtained, the characteristic absorption peak area ratio A (CA)/A (beta-CD) of the beta-cyclodextrin and the citric acid is plotted with a corresponding mass ratio M (CA)/M (beta-CD) standard curve, and the measured characteristic peak area ratio and the corresponding grafting rate of a synthetic product can be determined through the standard curve.

Has the advantages that: the invention adopts beta-cyclodextrin and citric acid as raw materials, immobilized lipase as a catalyst, the activity and the hydrophilicity of the product are further improved, the preparation method has the characteristics of green raw materials and catalysts, mild reaction temperature and high grafting rate, and the grafting rate test method is simple, convenient and effective. The citric acid-beta-cyclodextrin grafting compound prepared by the invention can be used as a carrier material for targeted drug delivery.

Drawings

FIG. 1 is a synthetic route for citric acid functionalized β -cyclodextrin;

FIG. 2A (CA)/A (. beta. -CD) -M (CA)/M (. beta. -CD) standard curve;

FIG. 3 FT-IR spectrum of the graft product β -CD-COOH;

FIG. 4 of the graft product beta-CD-COOH¹H-NMR spectrum;

FIG. 5 contact angle test chart of beta-cyclodextrin and graft product beta-CD-COOH.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1

Accurately weighing 3g of beta-cyclodextrin, adding the beta-cyclodextrin into a 25mL three-neck flask containing 8mL of dimethyl sulfoxide, weighing 1.739g of citric acid, slowly adding the citric acid into the flask, controlling the pH value of the solution within the range of 6-8, adding 10mg of immobilized lipase, heating to 44 ℃, reacting for 16 hours under the stirring at the speed of 1500r/min, pouring a product into the beaker after the reaction is finished, carrying out back dissolution by using a solution with the proportion of anhydrous ethanol/deionized water of 65/35, centrifuging, and drying the product in a vacuum drying oven at the temperature of 50 ℃ to obtain a solid powdery product beta-CD-COOH with the grafting rate of 22.2%;

example 2

Accurately weighing 3g of beta-cyclodextrin, adding the beta-cyclodextrin into a 25mL three-neck flask containing 8mL of dimethyl sulfoxide, weighing 1.739g of citric acid, slowly adding the citric acid into the flask, controlling the pH value of the solution within the range of 6-8, adding 4mg of immobilized lipase, heating to 47 ℃, reacting for 12 hours under the stirring at the speed of 1500r/min, pouring a product into the beaker after the reaction is finished, carrying out back dissolution by using a solution with the proportion of anhydrous ethanol/deionized water of 65/35, centrifuging, and drying the product in a vacuum drying oven at the temperature of 50 ℃ to obtain a solid powdery product beta-CD-COOH with the grafting rate of 9.1%;

example 3

Accurately weighing 3g of beta-cyclodextrin, adding the beta-cyclodextrin into a 25mL three-neck flask containing 8mL of dimethyl sulfoxide, weighing 1.739g of citric acid, slowly adding the citric acid into the flask, controlling the pH value of the solution within the range of 6-8, adding 7mg of immobilized lipase, heating to 50 ℃, reacting for 20 hours under the stirring at the speed of 1500r/min, pouring a product into the beaker after the reaction is finished, carrying out back dissolution by using a solution with the proportion of anhydrous ethanol/deionized water of 65/35, centrifuging, and drying the product in a vacuum drying oven at the temperature of 50 ℃ to obtain a solid powdery product beta-CD-COOH with the grafting rate of 11.7%;

example 4

Accurately weighing 3g of beta-cyclodextrin, adding the beta-cyclodextrin into a 25mL three-neck flask containing 8mL of dimethyl sulfoxide, weighing 1.739g of citric acid, slowly adding the citric acid into the flask, controlling the pH value of the solution within the range of 6-8, adding 13mg of immobilized lipase, heating to 53 ℃, reacting for 8 hours under the stirring at the speed of 1500r/min, pouring a product into the beaker after the reaction is finished, carrying out back dissolution by using a solution with the proportion of anhydrous ethanol/deionized water of 65/35, centrifuging, and drying the product in a vacuum drying oven at the temperature of 50 ℃ to obtain a solid powdery product beta-CD-COOH with the grafting rate of 19.6%;

FIG. 3 is a FT-IR spectrum, 3309cm, of a synthesized beta-cyclodextrin and citric acid grafted product beta-CD-COOH^-1The strong peak is-OH characteristic peak of beta-CD molecule, 3313cm^-1And 3232cm^-1Is a characteristic absorption peak of-OH at different positions in CA, and the characteristic absorption peak of-OH in beta-CD-COOH is formed from 3309cm^-1Moving to high wave number of 3402cm^-1This is due to the esterification of β -CD with CA. 1759 1691cm^-1Characteristic absorption peak of C ═ O in CA, 1742cm^-1Is a characteristic peak of ester carbonyl after esterification of CA and beta-CD, the beta-CD has no peak, and three curves of CA, beta-CD and beta-CD-COOH are 1119cm^-1、1187cm^-1And 1140cm^-1A characteristic peak of C-O-C is formed, and the beta-CD-COOH has a C-O-C bond, a characteristic peak of C ═ O bond and a shift of a characteristic peak of-OH bond as seen on a beta-CD-COOH curve, so to sayMin β -CD and CA form an ester.

FIG. 4 is a diagram of the synthesis of beta-cyclodextrin and citric acid grafted product beta-CD-COOH¹H-NMR spectrum, C ═ O appears to be electroattractive due to the binding of C ═ O structure to-OH in CA, unmasked, so that the shift of H-6 (Δ ═ 0.02ppm) at δ 3.55 shifts to high field, the H-1(J ═ 2.4Hz) peak at δ 4.83 splits into doublets by spin coupling, the shift peak of H-5 at δ 3.57 also appears to shift to low field (Δ ═ 0.02ppm), the hydrophobic interaction of the internal protons in H-5's masked β -CD, the protons are closer to the internal cavity of β -CD, while the protons on the remaining β -CD have no significant shift change.

FIG. 5 is a contact angle test chart of beta-cyclodextrin and a graft product beta-CD-COOH, wherein the beta-cyclodextrin contains a large number of hydroxyl groups, has an external hydrophilic structure and an internal hydrophobic structure, so that the beta-cyclodextrin has a strong hydrophilic property, and the citric acid contains three carboxyl groups and one hydroxyl group and also has a strong hydrophilic property. The water contact angle of the grafted product is smaller than the hydrolysis antenna of the raw material beta-cyclodextrin, which shows that the hydrophilicity of the grafted product is improved, and the grafted product is favorable for a carrier material for drug delivery.

The foregoing examples are provided for illustration and description of the invention only and are not intended to limit the invention to the scope of the described examples. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teachings of the present invention, which variations and modifications are within the scope of the present invention as claimed.