CN110183546B - 一种凝胶多糖水溶性抗菌衍生物及其制备方法 - Google Patents
一种凝胶多糖水溶性抗菌衍生物及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种凝胶多糖水溶性抗菌衍生物抗菌活性的研究,包括氨基化及季铵化凝胶多糖抗菌剂的合成和甜菜碱型季铵化凝胶多糖细胞毒性的评估,其中季铵盐与直接氨基改性的凝胶多糖或接枝赖氨酸的氨基凝胶多糖的摩尔比为5:1,甜菜碱型的季铵盐为两性物质,酸性基团以羧基为主,碱性基团以季铵盐为主;本发明还公开了上述包含凝胶多糖衍生物的抗菌活性和细胞毒性,本发明的产品具有良好的水溶性、生物相容性和显著的抗菌活性,尤其是对金黄色葡萄球菌的抗菌效果更为突出,氨基化及季铵化凝胶多糖的溶解性和抗菌性要优于凝胶多糖本身,并且不会破会凝胶多糖的骨架结构,提高了季铵盐的稳定性,为其作为抗菌材料的应用提供了基础。
Description
技术领域
本发明属于抗菌材料领域,涉及氨基化及季铵化改性后凝胶多糖的细胞毒性及抗菌活性的研究,具体为一种凝胶多糖抗菌衍生物及其制备方法。
背景技术
抗菌材料及抗菌剂在水消毒、纺织品、包装、建筑、医药以及食品领域等许多行业中都具有重要的研究意义,目前,虽然抗生素是控制细菌感染的主要措施,但由于常规抗生素的毒性以及细菌和真菌对抗生素的抗药性增加,导致致死性传染病的发病率增加,因此开发具有生物相容性和抗菌活性的抗菌剂是一种新的需求;近年来,各种抗菌药物如金属离子、金属纳米粒子、无机纳米材料、阳离子聚合物和抗菌肽等已用于预防病原体定殖,这为在细菌感染的治疗中取代抗生素的使用提供了新的可能性,由于大多数抗菌物质带正电荷,而细菌的细胞壁带负电,因此具有阳离子特性的多糖是值得探索的一类聚合物抗菌剂,可以有效地减少细菌和微生物的生长。
凝胶多糖是天然多糖的一种,作为一种可生物降解的天然生物材料,对人类和环境无毒、可食用,目前已在食品工业中作为胶凝剂和质地改性剂显示出实际的应用价值,并且由于其良好的生物活性使得凝胶多糖及其衍生物可用作药物递送的载体,在生物医药方面显示出了潜在的应用价值,但由于Curdlan分子间/分子内氢键导致的水不溶性限制了其应用,而凝胶多糖分子中存在大量的羟基,可在重复的羟基单元上引入不同的基团,改善凝胶多糖衍生物的溶解度,并可赋予其更多的生物活性,其中氨基化及季铵化改性是赋予多糖功能特性的有效手段。
季铵化合物是常用的阳离子表面活性剂和抗微生物剂,具有很强的抗菌活性,优良的细胞膜通透性、低毒性,可作为杀菌剂用于农业 、医药、抗菌等多个行业;甜菜碱(BET)是一种天然存在的甲基化氨基酸,具有抗炎、抗癌、抗菌、抗氧化和稳定心血管的功能,作为抗氧化剂,有助于细菌、真菌、藻类、植物和动物的渗透调节,由于其具有季铵盐的结构和羧酸盐的功能,可用于制备生物降解和生物相容的阳离子表面活性剂,季铵盐通常会提高聚合物表面正电荷的密度并使其产生抗菌作用,因此,在Curdlan链上引入季铵基可以扩大其在医药领域的应用范围,如药物输送、伤口敷料和抗菌涂层等。
天然抗菌剂和抗菌药物的需求在不断地增长,制备氨基化和季铵化凝胶多糖具有可期的现实意义,并且有望用于洗涤,化妆品,造纸,制药,抗菌塑料,抗菌薄膜,抗菌垫和纺织工业等,通过氨基化及季铵化修饰天然多糖使其抗菌能力得到改善,适用于制备具有生物医学应用特性的新型抗菌材料。
发明内容
本发明为解决公知技术中抗生素和无机抗菌剂存在的问题而提供了一种具有抗菌活性的氨基化及季铵盐修饰的凝胶多糖抗菌衍生物。
本发明公开了一种氨基化及季铵化改性的凝胶多糖抗菌衍生物及抗菌性能的研究,包括6-氨基凝胶多糖、赖氨酸修饰的氨基化凝胶多糖以及季铵化改性后的6-氨基凝胶多糖季铵盐和接枝懒氨酸的凝胶多糖季铵盐,该氨基化和季铵盐修饰的凝胶多糖衍生物不仅溶解性好而且具有明显的抗菌活性,其中季铵盐与6-氨基凝胶多糖或接枝懒氨酸的凝胶多糖的摩尔比为5:1,季铵盐为两性表面活性剂,其中酸性基以羧基为主,碱性基团以季铵盐为主。
氨基化凝胶多糖及季铵化改性凝胶多糖抗菌衍生物的抗菌性能研究,采用以下步骤:
所述季铵盐的结构式如下:
其中R1、R2和R3分别代表C1-C8的烷基基团,它们可以相同也可以不同,
所述的氨基化凝胶多糖及季铵化凝胶多糖抗菌衍生物的结构式如下:
附图说明
图1为本发明中合成6-氨基凝胶多糖及引入季铵盐过程中Cur(A)、Cur-N3(B)、Cur- NH2(C)、Cur-NH-Bet(D)四种物质的红外图谱;
图2为本发明中合成赖氨酸修饰的氨基凝胶多糖及引入季铵盐过程中Cur(A)、Cur-N3(B)、Cur- Lys(C)、Cur- Lys-Bet(D)四种物质的红外图谱;
图3、4为本发明中合成6-氨基凝胶多糖及季铵化后凝胶多糖的核磁共振图;
图5、6为本发明中合成接枝赖氨酸的凝胶多糖及季铵化后的核磁共振图;
图7为本发明中6-氨基凝胶多糖及引入季铵盐基团后对HEK293T细胞的毒性结果;
图8为本发明中接枝赖氨酸的凝胶多糖及引入季铵盐基团后对HEK293T细胞的毒性结果;
图9和图10分别为本发明中合成凝胶多糖抗菌衍生物过程中各物质对105CFU/mlL和108CFU/mlL大肠杆菌的杀菌效果图,图中的a、b、c、d、e分别代表Bet、Cur-NH2、Cur-NH-Bet、Cur-Lys和Cur-Lys-Bet;
图11和图12分别为本发明中合成凝胶多糖抗菌衍生物过程中各物质对105CFU/mlL和108CFU/mlL金黄色葡萄球菌的杀菌效果图,图中的a、b、c、d、e分别代表Bet、Cur-NH2、Cur-NH-Bet、Cur-Lys和Cur-Lys-Bet。
具体实施方式
为能进一步了解本发明的发明内容、特点及功效,列举以下实施例,并配合附图详细说明如下:
实施例1
一种凝胶多糖抗菌衍生物的制备及细胞毒性的研究,采用以下步骤(季铵盐以甜菜碱为例):
(1)称取500mg的凝胶多糖(curdlan)于反应瓶中,在80℃下加DMF搅溶解拌30分钟,氮气(N2)保护下加NaN3,反应4小时,室温下再加PPh3和CBr4,继续反应。反应结束后加无水甲醇沉淀,离心后收集沉淀,真空干燥后得到白色固体,即叠氮化凝胶多糖(Cur-N3)。
(2)将(1)合成的Cur-N3于60℃加DMSO溶解加NaBH4,反应结束后加适量无水乙醇沉淀,收集沉淀,透析3天,冷冻干燥得白色絮状固体即为6-氨基凝胶多糖(Cur-NH2)。
(3)合成PA-Lys:称2.00 g赖氨酸于反应瓶中加适量溶剂溶解后,冰浴下加1mol/L的NaOH反应0.5h,加5.54mL的二碳酸二叔丁酯,室温反应16h,洗涤分离提纯,真空干燥得Boc-lys,再向产物中加EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺)和NHS(N-羟基琥珀酰亚胺)和DIPEA其摩尔比为1:1.2:1.2:1.2,反应8h,洗涤分离提纯,真空干燥得Boc-lys-osu,继续向产物中加丙炔胺(PA)和DIPEA摩尔比为1:1.2:1.2,反应24h,洗涤分离提纯,真空干燥得PA-lys-Boc,最后再加DCM和TFA(v\v2:3)反应40min后减压除去溶剂,加无水***沉淀,倒出***,抽干即得PA-Lys。
(4)取(1)的产物Cur-N3加DMSO溶解,接着依次加入(3)的产物 PA-lys、CuBr2、抗坏血酸和DIPEA摩尔比为1:5:6:6:6,反应结束后装入截留分子量为3500透析袋透析72h,冻干得接枝赖氨酸的凝胶多糖(Cur-Lys)。
(5)称取0.500g的甜菜碱(Bet)加DMSO溶解,加EDC和NHS充分活化其羧基,再将其加到步骤(2)中得到的6-氨基凝胶多糖中其中Cur-NH2:Bet: EDC: NHS: DIPEA=1:5:6:6:6(摩尔比),反应完成后,透析将杂质除去,浓缩冻干后得氨基凝胶多糖季铵盐(Cur-NH-Bet)。
(6)取0.500g的甜菜碱(Bet)溶解,加EDC和NHS充分活化其羧基,再将其加到步骤(5)中得到的Cur-Lys中,其中Cur-Lys:Bet: EDC: NHS:DIPEA =1:5:6:6:6(摩尔比),反应完成后,透析除去杂质,冻干后得赖氨酸接枝凝胶多糖季铵盐(Cur-Lys-Bet)。
(7)将HEK293T细胞移到96孔板中,加入100μL的琼脂培养基,在37℃、5% CO2培养箱中孵育24h。以空白(不加材料,只加培养基)为对照,再将样品Bet,Cur-NH2,Cur-NH-Bet,Cur-Lys和Cur-Lys-Bet分别依次配置成200mg/mL,100mg/mL,50…1.56mg/mL,0.78mg/mL的浓度,每孔加入100微升的样品溶液。37℃、5% CO2培养箱中孵育24h,加入20μL1×MTT溶液,4h后,用美国的Filter Max F5 Multi-Mode Microplate Reader酶标仪在波长为570nmnm处读取每孔其相应的光密度吸光值,每个样品的同一浓度均设个3平行实验,GraphPadPrism软件作图并对实验结果进行分析。
(8)依次将样品Bet, Cur-NH2, Cur-NH-Bet, Cur-Lys和Cur-Lys-Bet加二次水配制成10mg / mL,9 mg / mL,8 mg / mL,7 mg / mL,6 mg / mL和5mg / mL的浓度,将不同浓度的样品分别对大肠杆菌 (E.coli)和金黄色葡萄球菌 (S.aureus)的杀菌效果进行评估,菌的浓度分别为 108CFU/mlL和105CFU/mlL,接触时间为3h,每个样品的同一浓度要平行实验三次,计算其杀菌率并用Origin作图。
(9)请参见图7、8、9、10,从图中可以看出随着样品浓度的增加和菌浓度的降低,季铵化凝胶多糖对大肠杆菌和金黄色葡萄球菌的杀菌率增加,且对金黄色葡萄球菌的杀菌效果要高于大肠杆菌,在菌浓度为105CFU/mlL时抗菌剂对不同菌种的杀菌率都在99%以上,图7和图8分别为各样品在105CFU/mlL和108CFU/mlL菌浓度的时对大肠杆菌的杀菌效果图,图9和图10是样品在105CFU/mlL和108CFU/mlL菌浓度时对金黄色葡萄球菌的杀菌效果,图中的a、b、c、d、e分别代表Bet、Cur-NH2、Cur-NH-Bet、Cur-Lys和Cur-Lys-Bet。图1和图2分别为合成氨基化及季铵化凝胶多糖的红外表征图,图1中的A、B、C、D分别代表Cur、Cur-N3、Cur-NH2和Cur-NH-Bet,图2中的A、B、C、D分别代表Cur、Cur-N3、Cur-Lys和Cur-Lys-Bet,分析图可知,化合物在3200-3500cm-1之间都出现的是凝胶多糖的羟基吸收峰,1040 cm-1的峰是凝胶多糖中糖苷键C-O-C的吸收峰,在2110.3cm-1处(-N3)基团的吸收峰,在1663.2cm-1,1566.9cm-1和1381.8cm-1处出现新的特征性基团-CO-NH-的吸收峰,归属于C=O伸缩振动(酰胺I),N - H弯曲振动(酰胺II)和C -N伸缩振动振动峰,且在2900 -3000cm-1处是甜菜碱的亚甲基的的吸收峰,在1469 cm−1处发现的峰是三甲基的特征带,证明了季铵盐的存在,结合图3 Cur-NH-Bet和图4 Cur-Lys-Bet的核磁共振分析可知成功合成了季铵化凝胶多糖抗菌衍生物;参见图5和图6是季铵化凝胶多糖对HEK293T细胞的毒性结果图,图5中的A,B,C,分别为Bet、Cur-NH2和Cur-NH-Bet随着样品浓度的增加细胞毒性增大,细胞的存活率降低,Cur-NH2细胞毒性较大Cur-NH-Bet的细胞毒性较低,有用于医用材料开发潜质;图6中的A,B,C,分别为Bet、Cur-Lys和Cur-Lys-Bet,从图中可以看出经过甜菜碱改性后的Cur-Lys-Bet的细胞毒性明显低于Cur-Lys,Cur-Lys-Bet在低浓度时对细胞基本无毒性,生物相容性较好,具有作为生物医用材料开发的价值,
实施例2
一种凝胶多糖抗菌衍生物的制备及细胞毒性的研究,采用以下步骤(季铵盐以甜菜碱为):
(1)与实施例1的步骤(1)相同;
(2)与实施例1的步骤(2)相同;
(3)与实施例1的步骤(3)相同;
(4)与实施例1的步骤(4)相同;
(5)称取0.500g的甜菜碱(Bet)加DMSO溶解,加EDC和NHS充分活化其羧基,再将其加到步骤(2)中得到的6-氨基凝胶多糖中其中Cur-NH2:Bet: EDC: NHS: DIPEA=1:5:6:6:7.5(摩尔比),反应完成后,透析将杂质除去,浓缩冻干后得氨基凝胶多糖季铵盐(Cur-NH-Bet);
(6)取0.500g的甜菜碱(Bet)溶解,加EDC和NHS充分活化其羧基,再将其加到步骤(5)中得到的Cur-Lys中,其中Cur-Lys:Bet: EDC: NHS:DIPEA =1:5:6:6:7.5(摩尔比),反应完成后,透析除去杂质,冻干后得赖氨酸接枝凝胶多糖季铵盐(Cur-Lys-Bet);
(7)与实施例1的步骤(7)相同;
(8)与实施例1的步骤(8)相同;
(9)所得的季铵化抗菌衍生物的细胞毒性结果和抗菌效果与实例1的(9)相符;
实施例3
一种凝胶多糖衍生物抗菌衍生物的制备及抗菌性能的研究;采用以下步骤
(1)与实施例1的步骤(1)相同;
(2)与实施例1的步骤(2)相同;
(3)与实施例1的步骤(3)相同;
(4)与实施例1的步骤(4)相同;
(5)与实施例1的步骤(6)相同;
(6)取0.500g的甜菜碱(Bet)溶解,加EDC和NHS充分活化其羧基,再将其加到步骤(5)中得到的Cur-Lys中,其中Cur-Lys:Bet: EDC: NHS:DIPEA =1:10:12:12:15(摩尔比),反应完成后,透析除去杂质,冻干后得赖氨酸接枝凝胶多糖季铵盐(Cur-Lys-Bet);
(7)将样品Bet,Cur-Lys和Cur-Lys-Bet分别依次配置成200mg/mL,100mg/mL,50…1.56mg/mL,0.78mg/mL的浓度,对HEK293T细胞的毒性进行评估,每个样品的同一浓度均设个3平行实验,GraphPad Prism软件作图并对实验结果进行分析;
(8)依次将样品Bet, Cur-Lys和Cur-Lys-Bet配制成10mg / mL,9 mg / mL,8 mg/ mL,7 mg / mL,6 mg / mL和5mg / mL的浓度,分别对大肠杆菌 (E.coli)和金黄色葡萄球菌 (S.aureus)的杀菌效果进行评估,菌的浓度分别为 108CFU/mlL和105CFU/mlL,接触时间为3h,每个样品的同一浓度要平行实验三次;
(9)所得的季铵化凝胶多糖抗菌衍生物的细胞毒性结果和抗菌效果与实例1的(9)相符;
实施例4
一种凝胶多糖衍生物的制备及抗菌性能的研究;采用以下步骤
(1)与实施例1的步骤(1)相同;
(2)与实施例1的步骤(2)相同;
(3)与实施例1的步骤(3)相同;
(4)与实施例1的步骤(4)相同;
(5)与实施例1的步骤(5)相同;
(6)与实施例1的步骤(6)相同;
(7)与实施例1的步骤(7)相同;
(8)依次将样品Bet, Cur-NH2, Cur-NH-Bet, Cur-Lys和Cur-Lys-Bet加二次水配制成10mg / mL,5mg / mL,2.5 mg / mL,1.25mg / mL,0.625mg / mL和0.312mg / mL的浓度,将不同浓度的样品分别对大肠杆菌 (E.coli)和金黄色葡萄球菌 (S.aureus)的杀菌效果进行评估,菌的浓度分别为 108CFU/mlL,接触时间为3h,每个样品的同一浓度要平行实验三次,计算其杀菌率并用Origin作图。
(9)所得的季铵化凝胶多糖抗菌衍生物的细胞毒性结果与实例1的(9)相符;但在样品浓度小于5mg / mL时,对大肠杆菌和金黄色葡萄球菌的抗菌效果不明显。
Claims (1)
1.一种凝胶多糖衍生物在制备抗大肠杆菌和金黄色葡萄球菌的抗菌剂中的应用,其特征在于所述的凝胶多糖衍生物由如下方法制备得到:
(1)制备叠氮化的凝胶多糖:称取凝胶多糖,加DMF加热搅拌溶解,之后加NaN3,继续在加热下条件下反应,然后冷却至室温依次加入三苯基膦和四溴化碳,室温下反应,最后将反应好的样品沉淀,收集沉淀,干燥后即得叠氮化凝胶多糖;
(2)将(1)所得的产物加硼氢化钠还原制得了氨基化凝胶多糖即6-氨基凝胶多糖Cur-NH2;
(3)或将(1)所得的产物与含端炔基的赖氨酸反应,制得一种接枝赖氨酸的氨基化凝胶多糖Cur-Lys。
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