CN107903322B - Method for preparing antibacterial collagen based on safrole epoxidation modification - Google Patents
Method for preparing antibacterial collagen based on safrole epoxidation modification Download PDFInfo
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- CN107903322B CN107903322B CN201710927960.6A CN201710927960A CN107903322B CN 107903322 B CN107903322 B CN 107903322B CN 201710927960 A CN201710927960 A CN 201710927960A CN 107903322 B CN107903322 B CN 107903322B
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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Abstract
The invention discloses a method for preparing antibacterial collagen based on safrole epoxidation modification. According to the method, a weak epoxidation reagent is used for modifying safrole, and then covalent reaction of an epoxy group and an amino group on a macromolecular side chain of collagen is utilized to covalently introduce the safrole into the collagen, so that a collagen substrate is endowed with good antibacterial activity. The collagen material obtained by the modification method has a long-acting antibacterial function, the triple helix structure of the collagen base material is not damaged, the inherent biocompatibility of the collagen base material can be completely reserved, and the collagen base material has a wide application prospect in the field of collagen base biomedical materials.
Description
Technical Field
The invention relates to a method for preparing antibacterial collagen based on safrole epoxidation modification, and belongs to the field of biomass materials.
Background
Collagen is the most important structural protein of connective tissues of higher vertebrates, widely exists in animal tissues in the forms of skin, bones, tendons, ligaments, nerves, blood vessels, teeth and the like, and plays roles of supporting organs and protecting organisms. Collagen, the most abundant protein in mammals, accounts for about 25-33% of the total protein in the body of the mammal. The collagen has special triple helix conformation, endows a plurality of excellent biological properties such as degradability, hemostatic property, histocompatibility and the like, so that the collagen is widely applied to tissue engineering such as skin substitutes, artificial cartilages, artificial blood vessels and the like, and various aspects such as medical health care, beauty and cosmetics, new functional materials and the like.
However, natural collagen is very vulnerable to microbial attack such as bacteria due to its own structural factors. During the growth and reproduction of the microorganisms, the collagen is subjected to enzymolysis to generate impurities, the quality of the collagen product is reduced, and the collagen is inactivated. In addition to this, the constant proliferation and metabolism of microorganisms leads to an increase in the endotoxin content of microorganisms in collagen preparations. Therefore, collagen-based products, especially collagen products for biomedical applications, must be treated to provide antimicrobial properties.
For a long time, it has proved to be an effective method to significantly improve the antibacterial and antiseptic properties of collagen products by adding small-molecule antibacterial agents (such as p-chloro-m-cresol, N-octyl isothiazolinone, paraben, etc.). However, this preservative strategy, which relies solely on the sustained dissolution of the antimicrobial agent, has the following drawbacks. First, the sustained dissolution antimicrobial mode necessarily lacks long-lasting efficacy. Secondly, many conventional antibacterial agents exhibit physiological toxicity to mammals while effectively killing harmful microorganisms such as bacteria, and the addition of such agents affects the inherent biocompatibility of natural collagen.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for preparing antibacterial collagen based on safrole epoxidation modification, which is characterized in that the method comprises the following process steps and conditions, and the parts of the used materials are all in parts by weight:
(1) performing epoxidation modification on safrole:
dissolving 12-20 parts of weak epoxidation reagent in 100-200 parts of solvent, adding 8-12 parts of safrole, and stirring at 20-30 ℃ for 12-24 hours; then washing the product with 2-7% alkali water solution for 3-5 times, and then washing with deionized water for 3-5 times; finally, drying the oil layer by anhydrous magnesium sulfate, filtering and then distilling under reduced pressure to obtain the epoxy safrole;
(2) epoxidized safrole graft modified collagen:
dissolving 10-20 parts of collagen in 0.2-0.5mol/L acetic acid aqueous solution, coating the mixture to form a film, and drying at 20-25 ℃ to constant weight; soaking the collagen membrane in water solution with pH of 7-9 and epoxy safrole concentration of 3-10%; and after 12-48 hours, taking out the collagen membrane, and washing the collagen membrane for 3-5 times by using deionized water to obtain the epoxidized safrole grafted and modified collagen.
In the above method, the weak epoxidation reagent in step (1) is one or more of m-chloroperoxybenzoic acid, iodosobenzene, and tert-butyl peroxide.
In the above method, the solvent in step (1) is one or more of dichloromethane, chloroform and carbon tetrachloride.
In the above method, the alkali in step (1) is one or more of sodium hydroxide, potassium hydroxide and sodium bicarbonate.
Compared with the prior art, the invention has the following positive effects:
1. according to the invention, the safrole is modified by adopting a weak epoxidation reagent, and the epoxidation reaction only acts on allyl in the safrole molecule and does not affect the remaining structure domain with the antibacterial effect, so that the antibacterial activity of the epoxidized and modified safrole is retained.
2. After the safrole is subjected to epoxidation modification, the epoxy group of the safrole can react with the amino group on the collagen macromolecular chain, so that the safrole is covalently fixed on the collagen material, and the antibacterial function of the collagen material has long-acting property.
3. The reaction conditions of the epoxidized safrole and the collagen are very mild, so that the antibacterial collagen prepared by adopting the technical route of the invention does not damage the inherent triple helix structure and biocompatibility of the collagen material.
Drawings
Fig. 1 is a preparation route of the epoxidized safrole according to the present invention.
Fig. 2 is a schematic diagram of the covalent reaction between the epoxidized safrole and collagen macromolecules according to the present invention.
Detailed Description
The invention is described in detail below with reference to examples, which are intended to be illustrative only and not to be construed as limiting the scope of the invention, and many insubstantial modifications and variations of the invention can be made by an engineer skilled in the art based on the teachings of the invention.
Example 1:
(1) performing epoxidation modification on safrole:
dissolving 12 parts of m-chloroperoxybenzoic acid in 100 parts of dichloromethane, adding 8 parts of safrole, and stirring at 20 ℃ for 24 hours; thereafter, the product was washed 3 times with 2% aqueous sodium hydroxide solution and then 3 times with deionized water; finally, drying the oil layer by anhydrous magnesium sulfate, filtering and then distilling under reduced pressure to obtain the epoxy safrole;
(2) epoxidized safrole graft modified collagen:
dissolving 10 parts of collagen in 0.2mol/L acetic acid aqueous solution, coating the mixture to form a film, and drying at 20 ℃ to constant weight; soaking the collagen membrane in water solution with pH of 7 and epoxidized safrole concentration of 3%; after 12 hours, the collagen membrane is taken out and washed by deionized water for 3 times to obtain the epoxidized safrole graft modified collagen.
The results of infrared spectroscopy and circular dichroism experiments show that the three-strand helical structure of the collagen is not damaged by the modification; toxicity experiments show that the LD50 value of the product is more than 55g/kg, the cytotoxicity grade is 0 grade (ISO10993-5), so the product can be regarded as safe and nontoxic; the modified collagen obtained by the method has the bacteriostasis rate of more than 90.2 percent on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa (a plate counting method).
Example 2:
(1) performing epoxidation modification on safrole:
dissolving 15 parts of iodosobenzene in 150 parts of chloroform, adding 10 parts of safrole, and stirring at 25 ℃ for 20 hours; thereafter, the product was washed 4 times with 5% aqueous potassium hydroxide solution and 5 times with deionized water; finally, drying the oil layer by anhydrous magnesium sulfate, filtering and then distilling under reduced pressure to obtain the epoxy safrole;
(2) epoxidized safrole graft modified collagen:
dissolving 15 parts of collagen in 0.4mol/L acetic acid aqueous solution, coating the mixture to form a film, and drying at 22 ℃ to constant weight; soaking the collagen membrane in water solution with pH of 8 and epoxidized safrole concentration of 7%; and after 24 hours, taking out the collagen membrane, and washing the collagen membrane for 4 times by using deionized water to obtain the epoxidized safrole grafted and modified collagen.
The results of infrared spectroscopy and circular dichroism experiments show that the three-strand helical structure of the collagen is not damaged by the modification; toxicity experiments show that the LD50 value of the product is more than 60g/kg, the cytotoxicity grade is 0 grade (ISO10993-5), so the product can be regarded as safe and nontoxic; the modified collagen obtained by the method has the bacteriostasis rate of more than 91.6 percent on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa (a plate counting method).
Example 3:
(1) performing epoxidation modification on safrole:
dissolving 20 parts of tert-butyl peroxide in 200 parts of carbon tetrachloride, adding 12 parts of safrole, and stirring at 30 ℃ for 12 hours; thereafter, the product was washed 5 times with 7% aqueous sodium bicarbonate solution and 5 times with deionized water; finally, drying the oil layer by anhydrous magnesium sulfate, filtering and then distilling under reduced pressure to obtain the epoxy safrole;
(2) epoxidized safrole graft modified collagen:
dissolving 20 parts of collagen in 0.5mol/L acetic acid aqueous solution, coating the mixture to form a film, and drying at 25 ℃ to constant weight; soaking the collagen membrane in an aqueous solution with the pH value of 9 and the concentration of the epoxidized safrole of 10 percent; and after 48 hours, taking out the collagen membrane, and washing the collagen membrane for 5 times by using deionized water to obtain the epoxidized safrole grafted and modified collagen.
The results of infrared spectroscopy and circular dichroism experiments show that the three-strand helical structure of the collagen is not damaged by the modification; toxicity experiments show that the LD50 value of the product is more than 60g/kg, the cytotoxicity grade is 0 grade (ISO10993-5), so the product can be regarded as safe and nontoxic; the modified collagen obtained by the method has the bacteriostasis rate of more than 91.2 percent on staphylococcus aureus, escherichia coli and pseudomonas aeruginosa (a plate counting method).
Claims (3)
1. A method for preparing antibacterial collagen based on safrole epoxidation modification is characterized in that the method comprises the following process steps and conditions, and the parts of the used materials are the parts by weight:
(1) performing epoxidation modification on safrole:
dissolving 12-20 parts of weak epoxidation reagent in 100-200 parts of solvent, adding 8-12 parts of safrole, and stirring at 20-30 ℃ for 12-24 hours; then washing the product with 2-7% alkali water solution for 3-5 times, and then washing with deionized water for 3-5 times; finally, drying the oil layer by anhydrous magnesium sulfate, filtering and then distilling under reduced pressure to obtain the epoxy safrole; the weak epoxidation reagent is one or more of m-chloroperoxybenzoic acid, iodosobenzene and tert-butyl peroxide;
(2) epoxidized safrole graft modified collagen:
dissolving 10-20 parts of collagen in 0.2-0.5mol/L acetic acid aqueous solution, coating the above collagen-acetic acid aqueous solution mixture to form a film, and drying at 20-25 deg.C to constant weight; soaking the collagen membrane in an aqueous solution prepared from the epoxidized safrole obtained in the step (1) and having a pH value of 7-9 and a concentration of 3-10%; and after 12-48 hours, taking out the collagen membrane, and washing the collagen membrane for 3-5 times by using deionized water to obtain the epoxidized safrole grafted and modified collagen.
2. The method for preparing antibacterial collagen according to claim 1, wherein the solvent in step (1) is one or more of dichloromethane, chloroform and carbon tetrachloride.
3. The method for preparing antibacterial collagen according to claim 1, wherein the alkali in step (1) is one or more of sodium hydroxide, potassium hydroxide and sodium bicarbonate.
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Citations (6)
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| WO2007042037A1 (en) * | 2005-10-07 | 2007-04-19 | Lifecycle Pharma A/S | Tacrolimus combination products |
| CN103505759A (en) * | 2013-07-04 | 2014-01-15 | 四川大学 | Method used for modifying collagen with epoxy quaternary ammonium salt |
| CN104013995A (en) * | 2014-06-26 | 2014-09-03 | 四川大学 | Oxidation chitosan graft modified porcine dermal collagen micro-nano fiber membrane and preparation method thereof |
| CN106349718A (en) * | 2016-10-25 | 2017-01-25 | 齐鲁工业大学 | Preparation method for antibacterial collagen |
| CN107011683A (en) * | 2017-04-17 | 2017-08-04 | 四川大学 | A kind of oxidation resistant edibility synthesized thin film of antibacterial and preparation method thereof |
| WO2017192923A1 (en) * | 2016-05-05 | 2017-11-09 | Monosol Rx, Llc | Pharmaceutical compositions with enhanced permeation |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007042037A1 (en) * | 2005-10-07 | 2007-04-19 | Lifecycle Pharma A/S | Tacrolimus combination products |
| CN103505759A (en) * | 2013-07-04 | 2014-01-15 | 四川大学 | Method used for modifying collagen with epoxy quaternary ammonium salt |
| CN104013995A (en) * | 2014-06-26 | 2014-09-03 | 四川大学 | Oxidation chitosan graft modified porcine dermal collagen micro-nano fiber membrane and preparation method thereof |
| WO2017192923A1 (en) * | 2016-05-05 | 2017-11-09 | Monosol Rx, Llc | Pharmaceutical compositions with enhanced permeation |
| CN106349718A (en) * | 2016-10-25 | 2017-01-25 | 齐鲁工业大学 | Preparation method for antibacterial collagen |
| CN107011683A (en) * | 2017-04-17 | 2017-08-04 | 四川大学 | A kind of oxidation resistant edibility synthesized thin film of antibacterial and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Collagen modified with epoxidized safrole for improving antibacterial activity;Zhou Xu;《The Royal Society of Chemistry》;20171027;第7卷;第50300–50306页 * |
| Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan;Dean-Hwa Shieh;《Oral Pathol Med》;20041231;第33卷;第581-587页 * |
| 芳樟油对大肠杆菌气相杀菌机制研究;吴克刚;《广东省食品学会第六次会员大会暨学术研讨会论文集》;20121231;第47-56页 * |
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