CN114712305B - In-situ dihydromyricetin-loaded BSA gel sterilization material, and preparation method and application thereof - Google Patents

In-situ dihydromyricetin-loaded BSA gel sterilization material, and preparation method and application thereof Download PDF

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CN114712305B
CN114712305B CN202210425222.2A CN202210425222A CN114712305B CN 114712305 B CN114712305 B CN 114712305B CN 202210425222 A CN202210425222 A CN 202210425222A CN 114712305 B CN114712305 B CN 114712305B
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dihydromyricetin
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CN114712305A (en
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阳明辉
陈翔
朱武
邓磊
夏恬恬
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Central South University
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Abstract

The invention discloses a BSA gel sterilization material loaded with dihydromyricetin in situ, and a preparation method and application thereof. The gel sterilization material is formed by uniformly loading active substances containing dihydromyricetin in a BSA gel material, and the gel sterilization material utilizes gel with a three-dimensional network structure constructed by BSA to load the dihydromyricetin with broad-spectrum sterilization performance in situ, is favorable for the slow release process of the dihydromyricetin, achieves the effects of long-term and continuous sterilization, has better biocompatibility, and can be widely applied to clinical preparations.

Description

In-situ dihydromyricetin-loaded BSA gel sterilization material, and preparation method and application thereof
Technical Field
The invention relates to a gel sterilization material, in particular to a BSA gel sterilization material loaded with dihydromyricetin in situ, and also relates to a preparation method of the gel sterilization material loaded with dihydromyricetin in situ and application of the gel sterilization material in preparation of sterilization agents, belonging to the technical field of biomedical materials.
Background
Bacterial infection is a major cause of various diseases in the human body, such as intact skin and mucous membrane are natural barriers preventing bacteria from invading the human body, from which bacteria are liable to invade the body after breakage. Dihydromyricetin is extracted from a woody vine plant of Ampelopsis genus of Vitaceae family, such as Ampelopsis grossedentata, has effects of scavenging free radicals, resisting oxidation, resisting thrombi, resisting tumor, diminishing inflammation, etc., especially has broad-spectrum antibacterial and bacteriostatic effects, and is a new generation of natural antibiotics, such as has obvious inhibition effect on common pathogenic bacteria in oral cavity and respiratory tract such as Staphylococcus aureus, staphylococcus epidermidis, beta-hemolytic streptococcus, escherichia coli, proteus vulgaris, pseudomonas aeruginosa, etc. However, dihydromyricetin is poor in water solubility and stability, resulting in limited application in clinical formulation.
Disclosure of Invention
Aiming at the problems of poor water solubility and stability and the like of the dihydromyricetin in the using process, the invention aims to provide the in-situ loaded dihydromyricetin BSA gel sterilization material, the active substances containing the dihydromyricetin are uniformly loaded in the BSA gel material, so that the solubility of the dihydromyricetin is improved, and the dihydromyricetin can be slowly released in the using process of the material because the dihydromyricetin is embedded in the gel material, so that the sterilization effect is realized, the long-term stability and effectiveness of the drug effect are realized, the dihydromyricetin contained in the gel sterilization material is derived from vine tea, the broad-spectrum antibacterial property is realized, the carrier material is derived from bovine serum, the structural similarity with human serum albumin is 76%, the biocompatibility of the gel sterilization material is improved, the BSA gel has better wetting property, and the drug effect of the dihydromyricetin is favorable to be exerted.
The second object of the invention is to provide a preparation method of a BSA gel sterilization material with simple operation, mild condition and low cost and in situ loading of dihydromyricetin, which takes BSA as a raw material, obtains three-dimensional network structure gel through reduction and oxidation reaction, and DMSO is not only used as an oxidant, but also used as a solvent of active substances containing dihydromyricetin, can improve the solubility of the dihydromyricetin, realize uniform dispersion and in situ loading of the dihydromyricetin in the gel, improve the sterilization performance of the gel material, simultaneously has higher glutathione content in human body as a reducing agent, has low DMSO biotoxicity as an oxidant and a solvent, can further improve the biocompatibility of the gel sterilization material, has simple operation, mild condition and low cost in the preparation process of the whole sterilization material, and is excellent in the performance of the obtained sterilization material, favorable for industrial production and has obvious advantages compared with the prior art.
The third purpose of the invention is to provide the application of the in-situ loading dihydromyricetin BSA gel sterilization material, and the application of the in-situ loading dihydromyricetin BSA gel sterilization material in preparation of a medicament with sterilization effect, wherein the obtained sterilization medicament can slowly release the active ingredient of the dihydromyricetin sterilization, has long-term stable and effective drug effect, and has better inactivation effect on common bacteria such as escherichia coli, staphylococcus aureus and the like.
In order to achieve the technical aim, the invention provides a BSA gel sterilization material loaded with dihydromyricetin in situ, which is formed by uniformly loading active substances containing dihydromyricetin into the BSA gel material.
The in-situ dihydromyricetin-loaded BSA gel sterilization material disclosed by the invention contains the dihydromyricetin active ingredient which is derived from vine tea, has broad-spectrum antibacterial property, and BSA is derived from bovine serum, and BSA and human serum albumin have 76% of similarity in structure, so that the gel sterilization material prepared by compounding the BSA and the human serum albumin has good biocompatibility, meanwhile, the solubility of the dihydromyricetin is improved, and the BSA gel has good wettability, so that the efficacy of the dihydromyricetin is exerted. Because the dihydromyricetin is embedded in the gel, the dihydromyricetin can be slowly released in the use process of the material, the sterilization effect is achieved, and the long-term stability and effectiveness of the drug effect are realized. Therefore, the dihydromyricetin is loaded in the BSA gel material, and the dihydromyricetin and the BSA gel material form obvious synergistic effect, so that on one hand, the solubility of the dihydromyricetin can be improved, the biocompatibility of the whole material is promoted, and on the other hand, the dihydromyricetin can be slowly released, and the long-term stable sterilization effect is realized.
As a preferred scheme, the BSA gel material is obtained by sequentially subjecting BSA to glutathione reduction and DMSO oxidation.
The BSA gel material adopts BSA, glutathione and biological low-toxicity DMSO as reaction raw materials, has good biocompatibility, can generate synergistic effect with dihydromyricetin, is beneficial to the exertion of the drug effect of the dihydromyricetin, realizes the slow release of the dihydromyricetin, and achieves the long-term stable sterilization effect.
As a preferred scheme, the active substance containing dihydromyricetin is dihydromyricetin and/or vine tea extract containing dihydromyricetin.
The BSA gel material can effectively load dihydromyricetin and ampelopsis grossedentata extract containing dihydromyricetin, and the dihydromyricetin and the ampelopsis grossedentata extract containing dihydromyricetin can be compounded with the BSA gel material to form the gel sterilization material with excellent performance.
As a preferable scheme, the content of dihydromyricetin in the active substance containing dihydromyricetin in the BSA gel material is 3-5 mg/mL.
The content of dihydromyricetin in the BSA gel material has a great influence on the material performance, and needs to be controlled within a certain range. If the content of dihydromyricetin in the BSA gel material is too low, the gel material cannot play an effective bactericidal role; if the dihydromyricetin content is too high, the gel material is liable to have a certain cytotoxicity.
The invention also provides a preparation method of the in-situ loading dihydromyricetin BSA gel sterilization material, which comprises the steps of adding glutathione into BSA solution to perform reduction reaction, and then adding DMSO solution containing active substances containing dihydromyricetin to perform oxidation reaction.
The invention adopts BSA as a carrier material, BSA molecules contain rich disulfide bonds, glutathione can reduce the disulfide bonds in the BSA to form free sulfhydryl groups, and DMSO has weak oxidizing property, and can oxidize the free sulfhydryl groups to regenerate disulfide bonds to form a three-dimensional network structure, so that the gel is prepared. In addition, compared with water, the solubility of the dihydromyricetin in DMSO is greatly improved, and the active substance containing the dihydromyricetin is dissolved in DMSO in advance, so that the dihydromyricetin can be embedded in gel while the DMSO reacts with BSA.
As a preferable scheme, the temperature of the reduction reaction is 40-50 ℃ and the reaction time is 3-6 h.
As a preferable scheme, the temperature of the oxidation reaction is 40-50 ℃ and the reaction time is 1-3 h.
Because BSA used in the invention belongs to protein substances, the BSA has certain requirements on temperature, and when the reaction temperature exceeds 60 ℃, protein denaturation is easy to cause, and starch protein fibers are formed; gel formation is not favored when the temperature is too low, whereas gel formation is favored at slightly higher temperatures of 40-50 c.
As a preferable scheme, the BSA solution has a mass percentage concentration of 30% -50%.
As a preferred embodiment, the glutathione is added to the BSA solution at a concentration of 30 to 50mM.
In the invention, BSA is a gel main body material, the gel formation is affected by too low concentration of BSA, and the gel is hardened by too high concentration. Glutathione is used as a reducing agent to reduce disulfide bonds of BSA into free sulfhydryl groups, if the concentration of glutathione is too low, BSA cannot be reduced sufficiently, but too high concentration of glutathione can lead to gel with certain cytotoxicity.
As a preferred embodiment, the volume ratio of DMSO to BSA solution is 1: 15-25. The concentration of dihydromyricetin in the DMSO is 80-100 mg/mL.
DMSO in the present invention has two effects, one is to oxidize the free thiol in glutathione-reduced BSA, and the other is to serve as a solvent for the active substance containing dihydromyricetin. If the amount of DMSO is too small, on the one hand BSA cannot be oxidized efficiently to form a gel, and on the other hand, a sufficient amount of active substance containing dihydromyricetin cannot be dissolved sufficiently because the solubility of dihydromyricetin in DMSO is 80 to 100mg/mL at the oxidation reaction temperature. However, too much DMSO will result in a gel that is somewhat cytotoxic. The dihydromyricetin serving as a sterilization component of the gel material is dissolved in DMSO and then is loaded to the BSA gel material, so that the concentration of the dihydromyricetin in the DMSO has certain requirements, the sterilization performance of the gel material is influenced by the too low concentration, and the dosage of the DMSO is increased due to the too high concentration, so that the gel has certain cytotoxicity.
The invention also provides an application of the in-situ dihydromyricetin-loaded BSA gel sterilization material in preparing a sterilization medicament.
The gel sterilization material has injectability, can be extracted through a needle tube, can be injected into a human body to realize different applications, has excellent sterilization effect, can slowly release dihydromyricetin, and has better inactivation effect on common bacteria such as escherichia coli, staphylococcus aureus and the like.
Compared with the prior art, the technical scheme of the invention has the advantages that:
(1) The BSA gel is used as a carrier material to load the dihydromyricetin, so that the solubility of the dihydromyricetin is improved, and the biocompatibility of the composite material is promoted;
(2) The dihydromyricetin is embedded in the gel, so that the dihydromyricetin can be slowly released, and the long-term and continuous sterilization effect is achieved;
(3) The gel is prepared by the reaction of DMSO and BSA for the first time, the DMSO is used as a weak oxidant and a broad-spectrum solvent at the same time, and dihydromyricetin can be loaded in situ and efficiently dispersed into the gel in the gel preparation process, so that the medicine loading in situ is realized and the medicine loading capacity is improved;
(4) The BSA-based gel sterilization material has the advantages of simple preparation method, mild condition and low cost, and is beneficial to large-scale production.
Drawings
Fig. 1 is a schematic diagram of the synthesis of the gel sterilization material of the present invention and a comparative diagram of the product synthesized under different conditions, wherein: (A) a synthetic schematic diagram of the gel sterilization material; (B) only BSA reacts with glutathione; (C) Adding DMSO (dimethyl sulfoxide) to prepare gel after BSA reacts with glutathione; (D) dissolving dihydromyricetin in DMSO.
FIG. 2 is a graph showing the slow release of dihydromyricetin by the gel sterilization material prepared in example 1.
Fig. 3 shows the sterilizing effect of the gel sterilization material and the control prepared in example 1 on escherichia coli and staphylococcus aureus, wherein the incubation of dihydromyricetin with bacterial solution, the incubation of pure bacterial solution, the incubation of DMSO with bacterial solution and the incubation of gel with bacterial solution are sequentially performed from left to right.
Fig. 4 shows the gel sterilization material prepared in example 1 injected through a syringe.
Detailed Description
The present invention will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the invention, but the scope of the invention is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present invention.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Example 1
An example of a gel sterilization material of the present invention is schematically shown in fig. 1. The specific preparation method of the gel sterilization material comprises the following steps:
(1) Glutathione (concentration of glutathione in the solution is 30 mM) was added to 5mL BSA solution (40 wt%) and the reduction reaction was carried out at 50℃for 5 hours;
(2) 1mL of the above solution was taken, and 50. Mu.L of DMSO solution containing 100mg/mL dihydromyricetin was added thereto to perform oxidation reaction at 50℃for 2 hours, thereby obtaining a gel antibacterial material.
The gel sterilization material prepared in example 1 was subjected to a dihydromyricetin release rate and release amount test, specifically, the gel and the buffer solution were incubated at room temperature, and the amount of dihydromyricetin released in the buffer solution was tested by ultraviolet spectrophotometry. As can be seen from FIG. 2, dihydromyricetin can be rapidly released from the gel within 12 hours, probably because the larger pores in the gel facilitate the release of dihydromyricetin. However, after 12 hours, the release speed of the dihydromyricetin is obviously slowed down, after 3 days, no dihydromyricetin is basically released, and after 5 days, the released dihydromyricetin reaches 65.91% of the loading capacity, which indicates that the gel sterilization material can realize the slow release of the dihydromyricetin, and the purpose of long-term stable sterilization is achieved.
Example 2
Another example of a gel sterilization material of the present invention is prepared by the following steps:
(1) Glutathione (concentration of glutathione in the solution was 50 mM) was added to 5mL of BSA (50 wt%) solution, and the reduction reaction was carried out at 40℃for 6 hours.
(2) 1mL of the above solution was taken, 50. Mu.L of DMSO solution containing 100mg/mL dihydromyricetin was added thereto, and oxidation reaction was carried out at 40℃for 3 hours to obtain a gel antibacterial material.
The gel sterilization material obtained by the preparation method of the embodiment shows obvious gel shape, and the gel performance is consistent with that of the gel material in the embodiment 1.
Example 3
The antibacterial performance of the gel antibacterial material prepared in example 1 was tested, specifically, the gel antibacterial material was incubated with a bacterial solution containing escherichia coli or staphylococcus aureus, and the antibacterial effect of the gel material was judged by observing the bacterial growth condition. Meanwhile, in order to better illustrate the sterilization performance of the gel material, three groups of control experiments are carried out, namely, pure bacteria incubation without any substances, dihydromyricetin incubation with bacteria liquid, DMSO incubation with bacteria liquid, and other operation conditions are kept unchanged.
As can be seen from FIG. 3, in the agar medium containing pure bacteria and DMSO, the E.coli and the Staphylococcus aureus grow vigorously, but in the agar medium containing the gel sterilization material or the dihydromyricetin, the E.coli and the Staphylococcus aureus do not obviously grow, which indicates that the gel sterilization material prepared by the method keeps the sterilization performance of the dihydromyricetin, shows excellent sterilization effect, and can be used as a sterilization material.
Example 4
An example of the application of the gel sterilization material of the present invention is that the gel has injectability, can be extracted through a needle tube, and can be injected into human bodies to realize different applications.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (8)

1. A BSA gel sterilization material loaded with dihydromyricetin in situ is characterized in that: the active substances containing dihydromyricetin are uniformly loaded in a BSA gel material;
the BSA gel material is obtained by sequentially reducing BSA by glutathione and oxidizing BSA by DMSO;
the content of dihydromyricetin in the BSA gel material is 3-5 mg/mL.
2. The in situ-supported dihydromyricetin BSA gel sterilization material according to claim 1, wherein the BSA gel sterilization material is characterized in that: the active substance containing dihydromyricetin is dihydromyricetin and/or vine tea extract containing dihydromyricetin.
3. The method for preparing the in-situ dihydromyricetin-loaded BSA gel sterilization material as claimed in claim 1 or 2, which is characterized by comprising the following steps: and (3) adding glutathione into the BSA solution to perform a reduction reaction, and then adding a DMSO solution containing active substances containing dihydromyricetin to perform an oxidation reaction to obtain the active substance.
4. The method for preparing the in-situ dihydromyricetin-loaded BSA gel sterilization material according to claim 3, wherein the method is characterized by comprising the following steps: the temperature of the reduction reaction is 40-50 ℃, and the reaction time is 3-6 h.
5. The method for preparing the in-situ dihydromyricetin-loaded BSA gel sterilization material according to claim 3, wherein the method is characterized by comprising the following steps: the temperature of the oxidation reaction is 40-50 ℃, and the reaction time is 1-3 h.
6. The method for preparing the in-situ dihydromyricetin-loaded BSA gel sterilization material according to claim 3, wherein the method is characterized by comprising the following steps:
the mass percentage concentration of the BSA solution is 30% -50%;
and the concentration of glutathione added into the BSA solution is 30-50 mM.
7. The method for preparing the in-situ dihydromyricetin-loaded BSA gel sterilization material according to claim 3, wherein the method is characterized by comprising the following steps:
the volume ratio of the DMSO solution to the BSA solution is 1:15-25;
the concentration of dihydromyricetin in the DMSO solution is 80-100 mg/mL.
8. Use of a BSA gel bactericidal material loaded with dihydromyricetin in situ as claimed in claim 1 or 2 for the preparation of a bactericidal medicament.
CN202210425222.2A 2022-04-22 2022-04-22 In-situ dihydromyricetin-loaded BSA gel sterilization material, and preparation method and application thereof Active CN114712305B (en)

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