CN113694042A

CN113694042A - Method for preparing cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning and application

Info

Publication number: CN113694042A
Application number: CN202110936479.XA
Authority: CN
Inventors: 宋梦; 常超康; 喻国敏; 秦琦; 岳秀琳; 曹凤仪; 焦书燕; 焦明立
Original assignee: Zhongyuan University of Technology
Current assignee: Zhongyuan University of Technology
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2021-11-26
Anticipated expiration: 2041-08-16
Also published as: CN113694042B

Abstract

The invention relates to a method for preparing a cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning and application thereof, wherein the method comprises the following steps: slowly adding cyclodextrin into a certain volume of solvent under stirring, stirring at room temperature until the cyclodextrin is completely dissolved, slowly adding flavonoid compound into the certain volume of solvent under stirring, stirring at a certain temperature until the flavonoid compound is completely dissolved, slowly adding the flavonoid compound solution into the cyclodextrin solution under stirring, stirring the mixed solution at the certain temperature for 8-12 hours, performing rotary evaporation, filtering to remove the non-included flavonoid compound, taking filtrate, freeze-drying to obtain an inclusion compound, and dissolving the inclusion compound in a small amount of deionized water to obtain a high-concentration spinning solution; and (3) carrying out electrostatic spinning by adopting the spinning solution to obtain a nanofiber antibacterial film, and finally carrying out vacuum drying to obtain the nanofiber antibacterial film. The method has the advantages of fewer steps, simplicity in operation, time saving and easiness in obtaining raw materials, can obtain the nano-scale antibacterial film, and is suitable for large-scale production.

Description

Method for preparing cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning and application

Technical Field

The invention belongs to the field of preparation of electrostatic spinning nanofiber membranes, and particularly relates to a method for preparing a cyclodextrin flavonoid inclusion compound nano antibacterial membrane by polymer-free electrostatic spinning and application of the cyclodextrin flavonoid inclusion compound nano antibacterial membrane.

Background

In recent years, electrostatic spinning technology is increasingly applied to the field of medicine, drugs can be distributed in nano fibers in an amorphous form through electrostatic spinning, the water solubility of the drugs is greatly increased to improve the bioavailability, and meanwhile, pharmacological properties of the drugs are not affected, so that the research on the application of electrostatic spinning to a drug delivery system has great application potential and good prospects. For example, a method for preparing beta-cyclodextrin inclusion compound electrospun fiber, such as julienne, grand bamboo, etc. (CN 102851789 a), prepares beta-cyclodextrin inclusion compound, adds polymer to form spinning solution, and carries out electrospinning to prepare nanofiber; shenwang, Sun Wei, etc. (CN 105568552A), a quercetin clathrate electrospinning nanomembrane, its preparation method and application use a solution method to prepare quercetin beta-cyclodextrin clathrate and use zein as polymer to prepare spinning solution for electrospinning to obtain a nanomembrane; neihua, Shuliju (CN 107287766A), a method for preparing polyacrylonitrile/curcumin nano-fiber mats by electrostatic spinning, directly prepares spinning solution added with polymers and carries out electrospinning to obtain the nano-mats. However, most of the research spinning solutions need to add polymers, and polymer-free electrostatic spinning can be performed without considering the types of polymers and the possible influence of the polymers, so that the operation steps are fewer, the operation is simpler and quicker, the materials are saved, and the cost is reduced.

Polymer-free electrospinning refers to a process in which a spinning solution without added polymer is jet-drawn into fibers, which can be prepared into nano-sized fibers. Compared with the traditional spinning method, the polymer-free electrostatic spinning nanofiber does not need to be added with a polymer, the equipment is simple, the operation is easy, the prepared nanofiber film has a mild drug loading mode, the drug activity cannot be damaged, and the drug is mostly distributed in the carrier in an amorphous form, so that the slow release of the drug is facilitated, and the bioavailability is effectively improved.

The flavonoid compound is a natural micromolecule compound, and modern pharmacological research shows that the flavonoid compound has wide pharmacological activities including oxidation resistance, antibiosis, anti-inflammation, reduction of vascular fragility, inhibition of tumor cells and the like. However, most of the flavonoid compounds are crystalline solids, are poor in water solubility and easy to degrade, phenolic hydroxyl groups in the structure are easy to oxidize, and the bioavailability is extremely low, so that the clinical application of the flavonoid compounds is limited. The nano-fiber antibacterial film is prepared by cyclodextrin inclusion and electrostatic spinning, so that the water solubility of the flavonoid compound can be greatly improved, the bioavailability is increased, the toxic and side effects can be reduced, and the safety of medication can be improved. The single drug film has the characteristic of quick dissolution, and can be used for administration modes such as oral administration, implantation, injection and the like. The composite material is compounded with other spinning films, is used for wound dressing, and has the advantages of drug sustained release, air permeability and the like.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for preparing a cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning, which is simple, rapid, easy to operate, high-efficiency and low in cost, is suitable for batch production, and provides reference for novel transdermal drug delivery or skin wound dressing.

In order to solve the technical problems, the invention adopts the following technical scheme:

a method for preparing cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning comprises the following steps:

(1) preparing a cyclodextrin flavonoid clathrate compound: the preparation method comprises the steps of preparing by a solution method, slowly adding cyclodextrin into deionized water under stirring, stirring at room temperature until the cyclodextrin is completely dissolved to obtain a cyclodextrin solution, slowly adding the flavonoid compound into absolute ethyl alcohol under stirring, stirring at 25-40 ℃ until the flavonoid compound is completely dissolved to obtain a flavonoid compound solution, slowly adding the flavonoid compound solution into the cyclodextrin solution under stirring, stirring the mixed solution at 55-65 ℃ for 8-12 hours, performing rotary evaporation, filtering to remove the non-included flavonoid compound, and freeze-drying the filtrate to obtain a cyclodextrin flavonoid inclusion compound;

(2) preparing a spinning solution: dissolving the cyclodextrin flavonoid inclusion compound obtained in the step (1) in deionized water to obtain a high-concentration spinning solution;

(3) and (3) performing electrostatic spinning on the spinning solution obtained in the step (2) to obtain a nanofiber antibacterial film, and finally performing vacuum drying to obtain the nanofiber antibacterial film.

Further, the cyclodextrin flavonoid inclusion compound prepared in the step (1) can be prepared by a grinding method, which comprises the following steps: adding deionized water into cyclodextrin, stirring to obtain paste, adding flavonoids, grinding, adding small amount of deionized water, filtering to remove non-included flavonoids, and freeze drying the filtrate.

Further, the ratio of the cyclodextrin to the flavonoid compound in the step (1) is 1:1 to 2: 1.

Further, no polymer is added in the preparation of the spinning solution in the step (2).

Further, the concentration of the high-concentration spinning solution in the step (2) is 160-200% (w/v).

And (3) electrostatic spinning is carried out by adopting an injector with the specification of 5mL, a needle with the specification of 22G-19G and the voltage of 15 kV-18 kV, a receiving screen adopts aluminum foil for grounding receiving, the distance between the needle and the receiving screen is 10-18 cm, the jet flow rate in the electrostatic spinning process is 0.3-1.0 mL/h, and the ambient humidity is 25% -50%.

The nanometer antibacterial film of the cyclodextrin flavonoid inclusion compound is prepared by the method.

The invention relates to application of a cyclodextrin flavonoid inclusion compound nano antibacterial film in the antibacterial field.

The method is simple, rapid and efficient, the raw materials are easy to obtain, and the nanofiber antibacterial film can be obtained.

Has the advantages that: 1. the method is simple and quick to operate, saves time, can obtain the nanofiber antibacterial film, and is suitable for large-scale production; 2. the raw materials used in the invention are cheap and easily available, and have application potential in the field of medicine. 3. The nanofiber antibacterial film prepared by the invention can be applied to transdermal drug delivery systems such as oral administration, implantation and injection or skin wound local dressings, and has wide application in the field of medicines. 4. When the nanofiber antibacterial film prepared by the invention is used in a transdermal drug delivery system, the effect of instant dissolution can be achieved, and the bioavailability is effectively improved; the composite material is compounded with other electrospun films (such as polylactic acid, gelatin, chitosan and the like) to form a double-layer or multi-layer dressing, so that the bacterial infection at the wound can be effectively avoided, and the wound healing is facilitated.

Drawings

Fig. 1 is an infrared spectrum of the 2-hydroxypropyl- β -cyclodextrin curcumin clathrate prepared in example 2;

fig. 2 is an X-ray diffraction pattern of the 2-hydroxypropyl- β -cyclodextrin curcumin inclusion complex prepared in example 2;

figure 3 is a differential scanning calorimetry plot of the 2-hydroxypropyl- β -cyclodextrin curcumin inclusion complex prepared in example 2;

fig. 4 is an electrospun film (a is the 2-hydroxypropyl- β -cyclodextrin curcumin inclusion electrospun film prepared in example 2, b is the 2-hydroxypropyl- β -cyclodextrin curcumin inclusion electrospun film prepared in comparative example 1, c is the 2-hydroxypropyl- β -cyclodextrin electrospun film);

FIG. 5 is a photograph of a sample before culturing Staphylococcus aureus (a-example 2, b-comparative example 1);

FIG. 6 is a photograph of a sample after culturing Staphylococcus aureus (a-example 2, b-comparative example 1);

FIG. 7 is a photograph of the dissolution of the electrospun film in water (left is the electrospun film of comparative example 1 and right is the electrospun film of example 2).

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1

(1) preparing a cyclodextrin curcumin clathrate compound: slowly adding 2-hydroxypropyl-beta-cyclodextrin into deionized water with stirring, stirring at room temperature until the mixture is completely dissolved, slowly adding curcumin with the amount of the cyclodextrin 1/2 substance into absolute ethyl alcohol with stirring, stirring at 30 ℃ until the curcumin is completely dissolved, slowly adding curcumin solution into cyclodextrin solution with stirring, stirring the mixed solution at 60 ℃ for 8 hours, performing rotary evaporation, filtering, taking filtrate, and freeze-drying to obtain an inclusion compound;

(2) preparing a spinning solution: dissolving the inclusion compound in the step (1) in 5mL of deionized water, standing for 2h, and preparing spinning solution with the inclusion compound concentration of 180% (w/v);

(3) using a 5 mL-specification injector and a needle head with the specification of 20G to extract the spinning solution obtained in the step (2), fixing the spinning solution on an electrostatic spinning device, carrying out electrospinning under the voltage of 15kV, the receiving distance of 10cm and the jet flow speed of 0.5mL/h to obtain a nanofiber antibacterial film;

(4) and putting the collected film into a vacuum drier for drying for 24h for later use.

Example 2

(1) preparing a cyclodextrin curcumin clathrate compound: adding cyclodextrin into deionized water, stirring to obtain paste, adding curcumin in an amount of the cyclodextrin 1/2 substance, grinding at room temperature for 2 hr, adding a small amount of deionized water, filtering, and freeze drying the filtrate;

(2) preparing a spinning solution: dissolving the inclusion compound in the step (1) in 5mL of deionized water, standing for 4h, and preparing into spinning solution with the concentration of 180% (w/v);

(3) extracting the spinning solution obtained in the step (2) by using a 5 mL-specification injector and a needle head with the specification of 19G, fixing the spinning solution on an electrostatic spinning device, carrying out electrospinning under the voltage of 15kV, the receiving distance of 18cm and the jet flow speed of 0.4mL/h to obtain a nanofiber antibacterial film;

FIG. 1 is an infrared spectrum of a cyclodextrin curcumin inclusion compound prepared in example 2; fig. 2 is an X-ray diffraction pattern of the cyclodextrin curcumin inclusion complex prepared in example 2; fig. 3 is a differential scanning calorimetry plot of the cyclodextrin curcumin inclusion prepared in example 2; the characteristic peak of CUR in FIG. 1 includes 3509.385cm due to stretching vibration of hydroxyl group^-1Absorption peak at position, 1628.135 cm generated by C = C stretching vibration^-1Absorption peak at 1602.582 cm^-1Stretching vibration of 1509.05 cm^-1Corresponds to a bending vibration of C = O, 962.3207cm^-1Peak of stretching vibration of C = O855.7712 cm^-1And is the peak of the out-of-plane bending vibration of C-H.

2-HP-β-CThe characteristic peak of D comprises 2924.086cm^-1Is CH₃Stretching vibration generation, 1631.51 cm^-1、1401.536 cm^-1The peak at corresponds to C-H₂1158.063 cm for telescopic vibration absorption^-1、1080.441 cm^-1、1027.408 cm^-1Is a C-O stretching vibration absorption peak.

In the physical mixture of 2-HP-beta-CD and CUR, the characteristic peaks of 2-HP-beta-CD and CUR are simultaneously appeared, and the characteristic peak of the CUR is 1628.135 cm^-1、1602.582 cm^-1、1510.014 cm^-1And 856.2534 cm^-1. The characteristic peak of the 2-HP-beta-CD appeared to be 1401.536 cm^-1、1154.688 cm^-1、1082.37 cm^-1、1027.408 cm^-1。

In the inclusion compound, the peak of CUR completely disappears and is covered by 2-HP-beta-CD, so that the spectrum of the inclusion compound is very similar to the infrared spectrum of 2-HP-beta-CD, which indicates that the inclusion of the CUR and the 2-HP-beta-CD in the inclusion compound is successful.

In figure 2, CUR is crystal powder, a plurality of sharp crystal diffraction peaks appear in an XRD diffraction pattern, 2-HP-beta-CD is amorphous powder, no crystal diffraction peaks exist in the pattern, the pattern of the mixture can be seen as the simple superposition of the patterns of HP-beta-CD and CUR, the crystal diffraction peak of CUR appears on the basis of the pattern of 2-HP-beta-CD, in the pattern of the inclusion compound, the crystal diffraction peak generated by the CUR completely disappears, the pattern is almost the same as the pattern of HP-beta-CD, and the situation that the pattern formed by the inclusion of the CUR molecules by the HP-beta-CD is amorphous is shown, so that the crystal diffraction peak does not appear any more.

In fig. 3, CUR has a sharp endothermic peak at around 176 °, corresponding to the melting point of curcumin, and in the physical mixture of β -CD and CUR, the peak generated by curcumin is slightly shifted and the intensity is significantly reduced, probably due to the inclusion reaction of a part of curcumin and cyclodextrin during the heating of the physical mixture. In the inclusion compound, the peak of CUR completely disappears, thus proving that a new substance is formed, namely, the inclusion of the inclusion compound is successful.

Comparative example 1

For comparison, a spinning solution was prepared directly on the basis of example 2, and the specific operations were as follows:

(1) preparing a spinning solution: adding cyclodextrin and curcumin with the mass ratio of 2:1 into 5mL of deionized water to enable the concentration of the solution to be 180% (w/v), stirring for 8h at 60 ℃, and cooling to room temperature to obtain spinning solution;

(2) extracting the spinning solution obtained in the step (2) by using a 5 mL-specification injector and a needle head with the specification of 19G, fixing the spinning solution on an electrostatic spinning device, carrying out electrospinning under the voltage of 15kV, the receiving distance of 18cm and the jet flow speed of 0.4mL/h to obtain a nanofiber antibacterial film;

(3) and putting the collected film into a vacuum drier for drying for 24h for later use.

In the antibacterial experiment, the diameter of a glass culture dish is 100mm, the culture dish is prepared with a soybean agar culture medium, the culture medium is uniformly coated with a staphylococcus aureus culture solution, two groups of 2-hydroxypropyl-beta-cyclodextrin curcumin inclusion compound electro-spinning films with the same mass are weighed (a sample in figure 4a is obtained by preparing the inclusion compound through grinding and electro-spinning, namely example 2, and a sample in figure 4b is obtained by directly preparing the electro-spinning solution and electro-spinning, namely comparative example 1), are trimmed and stacked into a square with the side length of 10mm, and the square is placed in the culture dish, so that the thickness of each part is not uniform due to stacking, the film is not uniformly diffused in the culture dish, as shown in figures 5a and 5b, the widest part 1 of the film in figure 5a is 22mm, the narrowest part 2 is 17mm, the widest part 1 of the film in figure 5b is 24mm, and the narrowest part 2 is 13mm after the film is diffused. After the membrane is cultured in a thermostat at 37 ℃ for 24 hours, as shown in fig. 6a and 6b, a dark transparent bacteriostatic ring is obviously formed around the cyclodextrin curcumin inclusion compound film, the average width of the bacteriostatic ring in fig. 6a and 6b is 2.5mm, and the culture medium outside the bacteriostatic ring is covered by staphylococcus aureus, so that the 2-hydroxypropyl-beta-cyclodextrin curcumin inclusion compound electrospun membrane has an inhibitory effect on staphylococcus aureus.

As shown in fig. 7, the electrospun film samples of comparative example 1 and example 2, which are weighed to be equal in mass, are respectively dissolved in water with equal amount, and it can be obviously observed that the upper layer of the solution of the comparative example 1 on the left side is suspended insoluble substances and is free curcumin; the sample in the sample solution of example 2 on the right side was completely dissolved and the solution was clear. Thus, an advantage of electrospinning with inclusion compounds in the examples is that there is no free curcumin in the film that is not included.

Comparative example 1 is compared with example 2, except that the two steps of preparing the cyclodextrin flavonoid inclusion compound and preparing the spinning solution in example 2 are simplified into one step. However, the comparative example 1 has the disadvantage that free curcumin exists on the nanofiber antibacterial film, the curcumin is difficult to dissolve and cannot be biologically utilized, and the embodiment has no problem, namely, the embodiment 2 has better antibacterial effect on the premise of the same curcumin dosage.

The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for preparing cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrostatic spinning is characterized by comprising the following steps:

(1) preparing a cyclodextrin flavonoid clathrate compound: the preparation method comprises the steps of preparing by a solution method, slowly adding cyclodextrin into deionized water under stirring, stirring at room temperature until the cyclodextrin is completely dissolved to obtain a cyclodextrin solution, slowly adding flavonoid compounds into absolute ethyl alcohol under stirring, stirring at 25-40 ℃ until the flavonoid compounds are completely dissolved to obtain a flavonoid compound solution, slowly adding the flavonoid compound solution into the cyclodextrin solution under stirring, stirring the mixed solution at 55-65 ℃ for 8-12 hours, performing rotary evaporation, filtering, and freeze-drying filtrate to obtain a cyclodextrin flavonoid inclusion compound;

2. The method for preparing the cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrospinning according to claim 1, wherein the method comprises the following steps: the cyclodextrin flavonoid inclusion compound prepared in the step (1) can be prepared by a grinding method, which comprises the following steps: adding deionized water into cyclodextrin, stirring to obtain paste, adding flavonoids, grinding, adding small amount of deionized water, filtering to remove non-included flavonoids, and freeze drying the filtrate.

3. The polymer-free electrostatic spinning method for preparing the cyclodextrin flavonoid inclusion compound nano antibacterial film according to claim 1 or 2, wherein the method comprises the following steps: the ratio of the cyclodextrin to the flavonoid compound in the step (1) is 1: 1-2: 1.

4. The method for preparing the cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrospinning according to claim 1, wherein the method comprises the following steps: and (3) no polymer is added in the preparation of the spinning solution in the step (2).

5. The method for preparing the cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrospinning according to claim 1, wherein the method comprises the following steps: the concentration of the high-concentration spinning solution in the step (2) is 160-200% (w/v).

6. The method for preparing the cyclodextrin flavonoid inclusion compound nano antibacterial film by polymer-free electrospinning according to claim 1, wherein the method comprises the following steps: and (3) electrostatic spinning is carried out by adopting an injector with the specification of 5mL, a needle with the specification of 22G-19G and the voltage of 15 kV-18 kV, a receiving screen adopts aluminum foil for grounding receiving, the distance between the needle and the receiving screen is 10-18 cm, the jet flow rate in the electrostatic spinning process is 0.3-1.0 mL/h, and the ambient humidity is 25% -50%.

7. The cyclodextrin flavonoid inclusion compound nano-antibacterial film prepared by the method of any one of claims 1-6.

8. The application of the cyclodextrin flavonoid inclusion compound nano-antibacterial film according to claim 7 in the antibacterial field.