CN107903322A - A kind of method that antibacterial collagen is prepared based on safrole epoxidation modification - Google Patents
A kind of method that antibacterial collagen is prepared based on safrole epoxidation modification Download PDFInfo
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- ZMQAAUBTXCXRIC-UHFFFAOYSA-N safrole Chemical compound C=CCC1=CC=C2OCOC2=C1 ZMQAAUBTXCXRIC-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 102000008186 Collagen Human genes 0.000 title claims abstract description 63
- 108010035532 Collagen Proteins 0.000 title claims abstract description 63
- 229920001436 collagen Polymers 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 24
- 238000006735 epoxidation reaction Methods 0.000 title claims abstract description 21
- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 20
- 230000004048 modification Effects 0.000 title claims abstract description 18
- 238000012986 modification Methods 0.000 title claims abstract description 18
- 239000000463 material Substances 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 9
- 229910021641 deionized water Inorganic materials 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 6
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000003756 stirring Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000003513 alkali Substances 0.000 claims description 4
- 239000000243 solution Substances 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 claims description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 3
- -1 tert-butanol peroxide Chemical class 0.000 claims description 3
- DKGAVHZHDRPRBM-UHFFFAOYSA-N tert-butyl alcohol Substances CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 claims description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims 3
- 125000001812 iodosyl group Chemical group O=I[*] 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 3
- 230000007774 longterm Effects 0.000 abstract description 3
- 125000003277 amino group Chemical group 0.000 abstract description 2
- 125000003700 epoxy group Chemical group 0.000 abstract description 2
- 229920002521 macromolecule Polymers 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract 2
- 238000002715 modification method Methods 0.000 abstract 1
- 244000005700 microbiome Species 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 238000004566 IR spectroscopy Methods 0.000 description 3
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 238000002983 circular dichroism Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 231100000820 toxicity test Toxicity 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- JYJVVHFRSFVEJM-UHFFFAOYSA-N iodosobenzene Chemical compound O=IC1=CC=CC=C1 JYJVVHFRSFVEJM-UHFFFAOYSA-N 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- ZHTRFSJGUKYTPR-UHFFFAOYSA-N 2-octyl-1,2-thiazolidin-4-one Chemical compound CCCCCCCCN1CC(=O)CS1 ZHTRFSJGUKYTPR-UHFFFAOYSA-N 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- CFKMVGJGLGKFKI-UHFFFAOYSA-N 4-chloro-m-cresol Chemical compound CC1=CC(O)=CC=C1Cl CFKMVGJGLGKFKI-UHFFFAOYSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 239000002473 artificial blood Substances 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 239000008204 material by function Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229940070805 p-chloro-m-cresol Drugs 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 210000000515 tooth Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- 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 or cold insoluble globulin [CIG]
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Abstract
本发明公开了一种基于黄樟素环氧化改性制备抗菌胶原的方法。该方法使用弱环氧化试剂改性黄樟素，再利用环氧基与胶原大分子侧链上氨基的共价反应，将黄樟素共价引入到胶原中，赋予胶原基材良好的抗菌活性。利用这种改性方法获得的胶原材料具有长效抗菌功能，胶原基材的三股螺旋结构并不会遭到破坏，其固有的生物相容性也能完整保留，在胶原基生物医用材料领域应用前景广阔。The invention discloses a method for preparing antibacterial collagen based on safrole epoxidation modification. The method uses a weak epoxidation reagent to modify the safrole, and then uses the covalent reaction between the epoxy group and the amino group on the side chain of the collagen macromolecule to covalently introduce the safrole into the collagen, so as to endow the collagen substrate with good antibacterial activity. The collagen material obtained by this modification method has a long-term antibacterial function, the triple helical structure of the collagen substrate will not be destroyed, and its inherent biocompatibility can also be completely preserved. It is applied in the field of collagen-based biomedical materials bright future.
Description
技术领域technical field
本发明涉及一种基于黄樟素环氧化改性制备抗菌胶原的方法，属于生物质材料领域。The invention relates to a method for preparing antibacterial collagen based on safrole epoxidation modification, which belongs to the field of biomass materials.
背景技术Background technique
胶原是高等脊椎动物结缔组织最主要的结构性蛋白，以皮肤、骨骼、肌腱、韧带、神经、血管、牙齿等形式广泛存在于动物组织中，起支撑器官及保护生物机体的作用。胶原作为哺乳动物体内含量最丰富的蛋白质，约占动物体内蛋白总量的25-33％。胶原所具有的特殊三股螺旋构象，赋予其许多优良的生物学性能，如可降解性、止血性和组织相容性等，从而让它在皮肤替代物、人工软骨和人工血管等组织工程以及各种医药保健、美容化妆、新功能材料等方面得到了广泛应用。Collagen is the most important structural protein in the connective tissue of higher vertebrates. It exists widely in animal tissues in the form of skin, bones, tendons, ligaments, nerves, blood vessels, and teeth, and plays a role in supporting organs and protecting biological organisms. Collagen is the most abundant protein in mammals, accounting for about 25-33% of the total protein in animals. The special triple-helix conformation of collagen endows it with many excellent biological properties, such as degradability, hemostasis, and tissue compatibility, so that it can be used in tissue engineering such as skin substitutes, artificial cartilage, and artificial blood vessels, as well as various It has been widely used in a variety of medicine and health care, beauty and makeup, and new functional materials.
然而，受自身结构因素的影响，天然胶原极易受到细菌等微生物侵蚀。微生物在生长繁殖的过程中，会酶解胶原产生杂质，降低胶原制品品质，使胶原蛋白失去活性。除此之外，微生物的不断增殖和代谢将导致胶原制品中微生物内毒素含量增加。因此，基于胶原蛋白的制品，尤其是应用于生物医药领域的胶原蛋白制品必须作抗菌处理。However, affected by its own structural factors, natural collagen is extremely susceptible to erosion by microorganisms such as bacteria. In the process of growth and reproduction, microorganisms will enzymatically decompose collagen to produce impurities, reduce the quality of collagen products, and make collagen inactive. In addition, the continuous proliferation and metabolism of microorganisms will lead to an increase in the content of microbial endotoxins in collagen products. Therefore, collagen-based products, especially collagen products used in the field of biomedicine must be treated with antibacterial treatment.
长期以来，通过外添加小分子抗菌剂(如对氯间甲酚、N-辛基异噻唑啉酮、尼泊金酯等)以显著提高胶原制品的抗菌防腐性被证明是一种行之有效的方法。然而，这种单纯依靠抗菌剂持续溶出的防腐策略存在如下缺陷。首先，持续溶出抗菌模式必然缺乏长效性。其次，许多传统抗菌剂在高效杀灭细菌等有害微生物的同时，也表现出对哺乳动物的生理毒性，其加入将影响天然胶原固有的生物相容性。For a long time, it has been proved to be an effective way to significantly improve the antibacterial and antiseptic properties of collagen products by adding small molecule antibacterial agents (such as p-chloro-m-cresol, N-octylisothiazolinone, paraben, etc.). Methods. However, this anti-corrosion strategy that relies solely on the continuous dissolution of antibacterial agents has the following defects. First of all, the continuous dissolution antibacterial mode inevitably lacks long-term effect. Secondly, while many traditional antibacterial agents can effectively kill harmful microorganisms such as bacteria, they also show physiological toxicity to mammals, and their addition will affect the inherent biocompatibility of natural collagen.
发明内容Contents of the invention
本发明的目的是为了克服现有技术存在的缺点和不足，而提供一种基于黄樟素环氧化改性制备抗菌胶原的方法，其特征在于该方法的工艺步骤和条件如下，所用物料的份数均为重量份数：The purpose of the present invention is to provide a kind of method for preparing antibacterial collagen based on safrole epoxidation modification in order to overcome the shortcomings and deficiencies in the prior art, which is characterized in that the process steps and conditions of the method are as follows, the number of parts of materials used Both are parts by weight:
(1)黄樟素环氧化改性：(1) Safrole epoxidation modification:
将12-20份弱环氧化试剂溶解于100-200份溶剂中，再加入8-12份黄樟素，于20-30℃搅拌12-24小时；此后，将产物用2-7％的碱水溶液洗涤3-5次，再用去离子水洗涤3-5次；最后，油层用无水硫酸镁干燥，过滤后减压蒸馏，得到环氧化黄樟素；Dissolve 12-20 parts of weak epoxidation reagent in 100-200 parts of solvent, then add 8-12 parts of safrole, and stir at 20-30°C for 12-24 hours; thereafter, the product is washed with 2-7% aqueous alkali solution washing 3-5 times, and then washing 3-5 times with deionized water; finally, the oil layer is dried with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain epoxidized safrole;
(2)环氧化黄樟素接枝改性胶原：(2) Epoxidized safrole grafted modified collagen:
将10-20份胶原溶于0.2-0.5mol/L的醋酸水溶液中，再将以上混合物涂覆成膜，于20-25℃干燥至恒重；再将以上胶原膜浸于pH值为7-9、环氧化黄樟素浓度为3-10％的水溶液中；12-48小时后，取出胶原膜，用去离子水清洗3-5次，获得环氧化黄樟素接枝改性胶原。Dissolve 10-20 parts of collagen in 0.2-0.5mol/L acetic acid aqueous solution, then coat the above mixture to form a film, and dry it at 20-25°C until it reaches constant weight; then soak the above collagen film in a pH value of 7- 9. Epoxidized safrole in an aqueous solution with a concentration of 3-10%. After 12-48 hours, take out the collagen film and wash it with deionized water for 3-5 times to obtain epoxidized safrole grafted modified collagen.
以上方法中，步骤(1)中所述弱环氧化试剂为间氯过氧苯甲酸、亚碘酰苯、过氧化叔丁醇中的一种或多种。In the above method, the weak epoxidation reagent described in step (1) is one or more of m-chloroperoxybenzoic acid, iodosobenzene, and tert-butanol peroxide.
以上方法中，步骤(1)中所述溶剂为二氯甲烷、氯仿、四氯化碳中的一种或多种。In the above method, the solvent described in step (1) is one or more of dichloromethane, chloroform, and carbon tetrachloride.
以上方法中，步骤(1)中所述碱为氢氧化钠、氢氧化钾、碳酸氢钠中的一种或多种。In the above method, the alkali described in step (1) is one or more of sodium hydroxide, potassium hydroxide and sodium bicarbonate.
本发明与现有技术相比，具有以下积极效果：Compared with the prior art, the present invention has the following positive effects:
1、本发明采用弱环氧化试剂对黄樟素进行改性，环氧化反应只作用于黄樟素分子中的烯丙基，并不会影响其剩余的、起抗菌作用的结构域，因此环氧化改性后黄樟素的抗菌活性得以保留。1, the present invention adopts weak epoxidation reagent to carry out modification to safrole, and epoxidation reaction only acts on the allyl group in safrole molecule, can not affect its remaining, the structural domain that plays antibacterial action, therefore epoxidation The antibacterial activity of safrole was preserved after modification.
2、黄樟素环氧化改性后，其环氧基可与胶原大分子链上的氨基发生反应，进而将黄樟素共价固定于胶原材料上，其抗菌功能具有长效性。2. After safrole is epoxidized and modified, its epoxy group can react with the amino group on the collagen macromolecular chain, and then covalently fix safrole on the collagen material, and its antibacterial function has long-term effect.
3、环氧化黄樟素与胶原的反应条件非常温和，因此，采用本发明的技术路线制备抗菌胶原，并不会破坏胶原材料固有的三股螺旋结构和生物相容性。3. The reaction conditions between epoxidized safrole and collagen are very mild, therefore, adopting the technical route of the present invention to prepare antibacterial collagen will not destroy the inherent triple helical structure and biocompatibility of the collagen material.
附图说明Description of drawings
图1为本发明所涉及的环氧化黄樟素制备路线。Fig. 1 is the preparation route of epoxidized safrole involved in the present invention.
图2为本发明所涉及的环氧化黄樟素与胶原大分子共价反应示意图。Fig. 2 is a schematic diagram of the covalent reaction between epoxidized safrole and collagen macromolecules involved in the present invention.
具体实施方式Detailed ways
下面通过实施例对本发明进行具体的描述，只用于对本发明进行进一步说明，不能理解为对本发明保护范围的限定，该领域的技术工程师可根据上述发明的内容对本发明作出一些非本质的改进和调整。The present invention is specifically described below by the embodiment, only for further illustrating the present invention, can not be interpreted as the limitation of protection scope of the present invention, the technical engineer of this field can make some non-essential improvements and improvements to the present invention according to the content of the above-mentioned invention Adjustment.
实施例1：Example 1:
(1)黄樟素环氧化改性：(1) Safrole epoxidation modification:
将12份间氯过氧苯甲酸溶解于100份二氯甲烷中，再加入8份黄樟素，于20℃搅拌24小时；此后，将产物用2％的氢氧化钠水溶液洗涤3次，再用去离子水洗涤3次；最后，油层用无水硫酸镁干燥，过滤后减压蒸馏，得到环氧化黄樟素；Dissolve 12 parts of m-chloroperoxybenzoic acid in 100 parts of dichloromethane, add 8 parts of safrole, and stir at 20°C for 24 hours; thereafter, the product is washed 3 times with 2% aqueous sodium hydroxide solution, and then used to remove Ionized water was washed 3 times; finally, the oil layer was dried with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain epoxidized safrole;
(2)环氧化黄樟素接枝改性胶原：(2) Epoxidized safrole grafted modified collagen:
将10份胶原溶于0.2mol/L的醋酸水溶液中，再将以上混合物涂覆成膜，于20℃干燥至恒重；再将以上胶原膜浸于pH值为7、环氧化黄樟素浓度为3％的水溶液中；12小时后，取出胶原膜，用去离子水清洗3次，获得环氧化黄樟素接枝改性胶原。Dissolve 10 parts of collagen in 0.2mol/L acetic acid aqueous solution, then coat the above mixture to form a film, and dry it at 20°C to constant weight; then soak the above collagen film in pH value 7, epoxidized safrole concentration 3% aqueous solution; after 12 hours, the collagen film was taken out and washed three times with deionized water to obtain epoxidized safrole grafted modified collagen.
红外光谱和圆二色谱实验结果显示，以上改性并没有破坏胶原蛋白的三股螺旋结构；毒性实验表明产物的LD50值大于55g/kg，细胞毒性等级为0级(ISO10993-5)，故产物可视为安全无毒；采用以上方法获得的改性胶原，对金黄色葡萄球菌、大肠杆菌、绿脓杆菌的抑菌率均＞90.2％(平板计数法)。The results of infrared spectroscopy and circular dichroism experiments show that the above modification does not destroy the triple helical structure of collagen; toxicity experiments show that the LD50 value of the product is greater than 55g/kg, and the cytotoxicity level is 0 (ISO10993-5), so the product can be It is regarded as safe and non-toxic; the modified collagen obtained by the above method has an antibacterial rate of >90.2% (plate count method) to Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
实施例2：Example 2:
(1)黄樟素环氧化改性：(1) Safrole epoxidation modification:
将15份亚碘酰苯溶解于150份氯仿中，再加入10份黄樟素，于25℃搅拌20小时；此后，将产物用5％的氢氧化钾水溶液洗涤4次，再用去离子水洗涤5次；最后，油层用无水硫酸镁干燥，过滤后减压蒸馏，得到环氧化黄樟素；Dissolve 15 parts of iodosobenzene in 150 parts of chloroform, add 10 parts of safrole, and stir for 20 hours at 25°C; after this, the product is washed 4 times with 5% potassium hydroxide aqueous solution, and then washed with deionized water for 5 time; at last, the oil layer is dried with anhydrous magnesium sulfate, and after filtration, vacuum distillation obtains epoxidized safrole;
(2)环氧化黄樟素接枝改性胶原：(2) Epoxidized safrole grafted modified collagen:
将15份胶原溶于0.4mol/L的醋酸水溶液中，再将以上混合物涂覆成膜，于22℃干燥至恒重；再将以上胶原膜浸于pH值为8、环氧化黄樟素浓度为7％的水溶液中；24小时后，取出胶原膜，用去离子水清洗4次，获得环氧化黄樟素接枝改性胶原。Dissolve 15 parts of collagen in 0.4 mol/L acetic acid aqueous solution, then coat the above mixture to form a film, and dry it at 22°C to constant weight; then soak the above collagen film in pH 8, epoxidized safrole concentration 7% aqueous solution; after 24 hours, the collagen film was taken out and washed 4 times with deionized water to obtain epoxidized safrole grafted modified collagen.
红外光谱和圆二色谱实验结果显示，以上改性并没有破坏胶原蛋白的三股螺旋结构；毒性实验表明产物的LD50值大于60g/kg，细胞毒性等级为0级(ISO10993-5)，故产物可视为安全无毒；采用以上方法获得的改性胶原，对金黄色葡萄球菌、大肠杆菌、绿脓杆菌的抑菌率均＞91.6％(平板计数法)。The results of infrared spectroscopy and circular dichroism experiments show that the above modification does not destroy the triple helical structure of collagen; toxicity experiments show that the LD50 value of the product is greater than 60g/kg, and the cytotoxicity level is 0 (ISO10993-5), so the product can be It is regarded as safe and non-toxic; the modified collagen obtained by the above method has an antibacterial rate of >91.6% (plate count method) to Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
实施例3：Example 3:
(1)黄樟素环氧化改性：(1) Safrole epoxidation modification:
将20份过氧化叔丁醇溶解于200份四氯化碳中，再加入12份黄樟素，于30℃搅拌12小时；此后，将产物用7％的碳酸氢钠水溶液洗涤5次，再用去离子水洗涤5次；最后，油层用无水硫酸镁干燥，过滤后减压蒸馏，得到环氧化黄樟素；Dissolve 20 parts of tert-butanol peroxide in 200 parts of carbon tetrachloride, add 12 parts of safrole, and stir at 30° C. for 12 hours; after this, the product is washed 5 times with 7% aqueous sodium bicarbonate solution, and then used to remove Washing with deionized water for 5 times; finally, the oil layer was dried with anhydrous magnesium sulfate, filtered and distilled under reduced pressure to obtain epoxidized safrole;
(2)环氧化黄樟素接枝改性胶原：(2) Epoxidized safrole grafted modified collagen:
将20份胶原溶于0.5mol/L的醋酸水溶液中，再将以上混合物涂覆成膜，于25℃干燥至恒重；再将以上胶原膜浸于pH值为9、环氧化黄樟素浓度为10％的水溶液中；48小时后，取出胶原膜，用去离子水清洗5次，获得环氧化黄樟素接枝改性胶原。Dissolve 20 parts of collagen in 0.5 mol/L acetic acid aqueous solution, then coat the above mixture to form a film, and dry it at 25°C to constant weight; then soak the above collagen film in pH 9, epoxidized safrole concentration 10% aqueous solution; after 48 hours, the collagen film was taken out and washed 5 times with deionized water to obtain epoxidized safrole grafted modified collagen.
红外光谱和圆二色谱实验结果显示，以上改性并没有破坏胶原蛋白的三股螺旋结构；毒性实验表明产物的LD50值大于60g/kg，细胞毒性等级为0级(ISO10993-5)，故产物可视为安全无毒；采用以上方法获得的改性胶原，对金黄色葡萄球菌、大肠杆菌、绿脓杆菌的抑菌率均＞91.2％(平板计数法)。The results of infrared spectroscopy and circular dichroism experiments show that the above modification does not destroy the triple helical structure of collagen; toxicity experiments show that the LD50 value of the product is greater than 60g/kg, and the cytotoxicity level is 0 (ISO10993-5), so the product can be It is regarded as safe and non-toxic; the modified collagen obtained by the above method has an antibacterial rate of >91.2% (plate count method) to Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.
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