CN116355114A - 一种鲟鱼硫酸软骨素的制备方法及其在促糖尿病慢性伤口愈合敷料中的应用 - Google Patents
一种鲟鱼硫酸软骨素的制备方法及其在促糖尿病慢性伤口愈合敷料中的应用 Download PDFInfo
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- CN116355114A CN116355114A CN202310161700.8A CN202310161700A CN116355114A CN 116355114 A CN116355114 A CN 116355114A CN 202310161700 A CN202310161700 A CN 202310161700A CN 116355114 A CN116355114 A CN 116355114A
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- chondroitin sulfate
- sturgeon
- carboxymethyl chitosan
- dressing
- cartilage
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Abstract
本发明涉及硫酸软骨素提取技术领域,为解决现有技术下硫酸软骨素的提取纯化过程复杂,需要使用大量化学试剂且对硫酸软骨素损失较大的问题,公开了一种鲟鱼硫酸软骨素的制备方法,包括将鲟鱼软骨颗粒热液化处理后进行酶解得到酶解液;将酶解液过滤纯化得到鲟鱼硫酸软骨素溶液。该方法化学试剂用量少,步骤简便,且对硫酸软骨素提纯的回收率高,可实现对硫酸软骨素的绿色工业化生产,同时可获得软骨胶原蛋白生物活性肽副产品。本发明还提供了一种含有鲟鱼硫酸软骨素的促糖尿病慢性伤口愈合敷料的应用,该敷料具备良好的止血抗菌性能、生物相容性、生物可降解性、组织粘附性能和机械性能,可促进糖尿病慢性伤口愈合,具有广泛的应用前景。
Description
技术领域
本发明涉及硫酸软骨素提取技术领域,尤其涉及一种鲟鱼硫酸软骨素的制备方法及其在促糖尿病慢性伤口愈合敷料中的应用。
背景技术
皮肤是保护人体免受外部细菌和病毒侵害的第一道防线,其完整性对人体安全极其重要。糖尿病溃疡是一类无序、久治不愈的慢性伤口,极大的增加了患者生活痛苦。据国际糖尿病联盟(IDF)发布的最新全球糖尿病地图(IDF Diabetes Atlas)(第九版)显示,预计到2045年,糖尿病患者将达到6.93亿。糖尿病患者发生溃疡的概率为15%~25%,有66%的患者将在治愈5年后会复发溃疡,12%的患者在溃疡复发后截肢,而截肢患者中的70%是因为溃疡没有得到及时有效的治疗。因此,亟需进一步开展针对干预糖尿病慢性伤口愈合敷料的制备。
天然硫酸软骨素以其独特的生物相容性、生物可降解性优势和本身具备抗菌活性、巨噬细胞调节活性等潜力在医用敷料领域受到广泛关注。硫酸软骨素(chondroitinsulfate,CS)是一类糖胺聚糖,富含硫酸基和羧基,且带有强负电荷,主要存在于人和动物的软骨、韧带、肌腱和动脉血管壁等组织的基质中。关于从软骨中分离硫酸软骨素的工业生产流程已经实行多年,一般包括以下四个步骤:(1)软骨的化学水解;(2)糖胺聚糖与核心蛋白的分解;(3)蛋白质的清除和硫酸软骨素的收集;(4)硫酸软骨素的纯化。前两个阶段主要是在含有高浓度氢氧化钠、尿素、半胱氨酸或盐酸胍的碱性溶液中进行,然后通过阳离子季铵盐、非离子洗涤剂、硫氰酸钾或酒精溶液选择性沉淀糖胺聚糖,通过三氯乙酸可以除蛋白质,最后用层析法纯化,得到符合纯度的硫酸软骨素。然而,传统硫酸软骨素提取工艺中通常涉及高浓度的碱、尿素、盐酸胍等化学物质,虽然会促进软骨的水解以及糖胺聚糖与核心蛋白的分离,但也会加剧环境污染。为了进一步追求可持续性,一些研究如公告号为CN102190740A、CN104017108A的发明专利,提出稀碱-酶解法、机械化学辅助萃取法等多种方法来代替传统工艺,主要步骤为:(1)加碱调节后添加生物酶水解软骨和蛋白质;(2)使用酒精溶液选择性沉淀硫酸软骨素;(3)中和反应溶液;(4)采用超滤-透析技术进行分子量分离进而纯化硫酸软骨素。其中,常用的水解蛋白酶有木瓜蛋白酶、碱性蛋白酶、胰蛋白酶、胃蛋白酶、枯草杆菌素酶和多种酶复配使用等。现有提取方法一定程度上减少了环境污染,但仍存在化学试剂的使用、纯化过程复杂等问题,造成大量硫酸软骨素的损失及工业化生产困难现状。
发明内容
本发明为了克服现有技术下硫酸软骨素的提取纯化过程复杂,需要使用大量化学试剂且对硫酸软骨素的损失较大的问题,提供一种鲟鱼硫酸软骨素的制备方法,该制备方法对硫酸软骨素的提取过程高效无损,总体操作简单适合工业化生产,绿色污染少符合当下可持续发展概念。本发明还提供了一种含有鲟鱼硫酸软骨素的促糖尿病慢性伤口愈合敷料,该敷料具备良好的止血抗菌性能、生物相容性、生物可降解性、组织粘附性能和机械性能,可促进糖尿病慢性伤口愈合,具有极高的产业化应用前景。
为了实现上述目的,本发明采用以下技术方案:
一种鲟鱼硫酸软骨素的制备方法,包括如下步骤:
(1)将鲟鱼软骨颗粒热液化处理;
(2)向处理后的鲟鱼软骨颗粒加入水和蛋白酶进行酶解,待酶解完全后加热灭酶得到酶解液;(3)将酶解液过滤纯化得到鲟鱼硫酸软骨素溶液。
硫酸软骨素的提取原料基本为传统陆生动物,陆生动物具有一定安全隐患,如疯牛病、猪流感、口蹄疫、禽流感等传染病。因此,水产原料来源的硫酸软骨素具有极大优势。鲟鱼(Sturgeon)是目前生活在地球上最古老、最原始、体型最大、寿命最长的软骨鱼种之一。我国为世界上鲟鱼养殖产量最大的国家,从2000年初的1.10万吨增长到2021年10.80万吨。鲟鱼鱼子酱为鲟鱼主要产品,具有极高经济价值,鲟鱼软骨作为加工副产物,其利用程度较低,产品结构单一、附加值少。本发明选用鲟鱼软骨作为硫酸软骨素制备原料,具有极高的产业化应用前景。将鲟鱼软骨粉碎后热液化处理或是蒸汽爆破处理,均可有效地破坏鱼骨的结构使其发生液化,可促进糖苷键的断裂并提高后续酶解效率,酶解前无需调整pH,化学试剂用量少且可省去后续中和步骤。本发明制备得到的鲟鱼硫酸软骨素与市面上鲨鱼硫酸软骨素(Shark-CS)相比,除物种多样性导致的差异外其余结构基本一致,也与稀碱-酶解-化学沉淀法从鲟鱼软骨中分离得到的硫酸软骨素相似。该制备过程得到的硫酸软骨素回收率高,还可同时获得鲟鱼软骨胶原蛋白肽。
作为优选,所述步骤(1)中鲟鱼软骨颗粒由将新鲜鲟鱼软骨煮至骨肉分离后打碎得到,或由干制鲟鱼软骨与水混合至固液比为1:(3~4)后打碎得到。
作为优选,所述步骤(1)的热液化处理为将鲟鱼软骨颗粒进行热液化,热液化条件为压力0.04~0.10MPa、温度105~115℃、时间1.5~2.5h。
热液化过程极易受美拉德反应影响,因此热液化温度不能过高。
作为优选,所述步骤(3)中蛋白酶为复合中性蛋白酶,由质量配比为(1~2):(1~2):(2~3)的风味蛋白酶、中性蛋白酶和木瓜蛋白酶组成,蛋白酶的添加量为0.2~0.8%。
作为优选,所述步骤(3)中,处理后的鲟鱼软骨颗粒和水的料液比为1:(3~6),酶解时温度为45~60℃,酶解时间为6~9h;加热至90~100℃后保温10~20min灭酶。
作为优选,所述步骤(4)中过滤纯化过程为将酶解液依次通过陶瓷膜过滤、RO反渗透膜浓缩、10kDa卷式膜超滤分离。
一种促糖尿病慢性伤口愈合敷料的制备方法,包括如下步骤:
1)将鲟鱼硫酸软骨素溶液经冻干、复溶后,再加入可溶性钙盐和羧甲基壳聚糖交联,得到鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖复合物填料;
2)将羧甲基壳聚糖冻融1~6次后,冻干得到羧甲基壳聚糖海绵;
3)将羧甲基壳聚糖海绵与填料混合得到鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖/羧甲基壳聚糖复合海绵水凝胶敷料。
鲟鱼硫酸软骨素具有促进成纤维细胞增殖、迁移、激活巨噬细胞、上调CD31、VEGF和PCNA的表达等作用,可促进表皮和真皮增生、血管再生,加速皮肤创面愈合。本发明中鲟鱼硫酸软骨素在Ca2+作用下交联,再基于席夫碱反应和羧甲基壳聚糖里面的氨基结合生成双键制得鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖复合物,将该复合物作为填料填充至羧甲基壳聚糖海绵中,获得鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖/羧甲基壳聚糖复合海绵水凝胶敷料。其中,填料中的Ca2+可作为凝血因子释放激活了参与级联凝血的主动方式;羧甲基壳聚糖的添加进一步增强了水凝胶的抗菌活性及机械稳定性。
作为优选,所述步骤1)中鲟鱼硫酸软骨素和可溶性钙盐中钙离子的摩尔比为(1~2):(3~5),鲟鱼硫酸软骨素和羧甲基壳聚糖的质量比为(0.5~1):1。
作为优选,所述步骤2)的冻融过程为将羧甲基壳聚糖溶解后-10~-20℃低温冷冻,再-30~-60℃超低温冷冻,最后冻干。
作为优选,所述步骤3)中羧甲基壳聚糖海绵与填料的质量比为1:(1~3)。
因此,本发明具有如下有益效果:(1)鲟鱼硫酸软骨素的制备过程中化学试剂用量少,对环境友好,步骤简便,且对硫酸软骨素的损耗少,提纯的回收率高,可实现对硫酸软骨素的绿色工业化生产,同时可获得软骨胶原蛋白生物活性肽副产品;(2)选用鲟鱼软骨作为硫酸软骨素制备原料,来源广泛成本低,生物安全隐患低;(3)将鲟鱼硫酸软骨素与羧甲基壳聚糖结合得到促糖尿病慢性伤口愈合敷料,该敷料具有良好的抗菌活性及机械稳定性,用于糖尿病病人慢性伤口的治疗时,可避免伤口感染并加速创面愈合。
附图说明
图1是硫酸软骨素结构图。
图2是本发明实施例1和对比例1-2所得软骨颗粒的微观结构图。
图3是L929细胞的迁移活性变化图。
图4是RAW264.7细胞的形态变化图。
图5是小鼠伤口的愈合影响及热图。
图6是伤口组织切片的免疫组化染色图。
具体实施方式
下面结合附图及具体实施方法对本发明做进一步的描述。
实施例1
一种鲟鱼硫酸软骨素的制备方法,步骤如下:
(1)鲟鱼软骨分割:将新鲜鲟鱼软骨加入水中加热,煮至骨肉分离,取出,打碎至颗粒状得到鲟鱼软骨颗粒备用;
(2)热液化技术(HP)前处理:将步骤(1)获得的鲟鱼软骨颗粒放置于高压反应釜中,加热至温度110℃,保温2h得到热液化软骨液,该条件下获得热液化鱼骨液的亮度值为65;(3)酶解:将步骤(2)获得的热液化软骨液按料液比1:4加水稀释,加入复合中性蛋白酶进行酶解,酶解条件为:温度55℃、时间9h、酶添加量0.2%;待酶解完全后,于100℃、10min条件下灭酶得到酶解液,该条件下蛋白的水解度为29%;
(4)超滤分离:将步骤(3)获得的酶解液依次通过陶瓷膜过滤、RO反渗透膜浓缩、10kDa卷式膜超滤分离得到鲟鱼硫酸软骨素溶液。
分别依次使用液相色谱联用十八角度激光散射仪和示差检测器(HPSEC-MALLS-RI)、Folin-酚法、硫酸-咔唑法、明胶比浊法和高效液相色谱法对实施例1所得鲟鱼软骨素进行检测。
实施例1获得的硫酸软骨素平均分子量为56.67±0.4kDa,蛋白质含量为9.66±1.3%,糖醛酸含量为29.60±0.44%,硫酸根含量为13.11±3.2%,单糖由葡萄糖醛酸(GlcA,39.88%)、氨基半乳糖(GalNAc,32.96%)、半乳糖(Gal,18.64%)和葡萄糖胺(GlcNAc,8.53%)组成,二糖由ΔDi6S(58.38%)、ΔDi4S(27.34)和ΔDi0S(14.29%)组成,证明所提取的多糖为硫酸软骨素。并且实施例1所得硫酸软骨素中组成主要为C型(CS-C)二糖单元,其次为A型二糖单元(CS-A)、O型二糖单元(CS-O),硫酸根的位置在乙酰氨基半乳糖的6位或者4位上,与图1硫酸软骨素的化学结构对比可证明实施例1得到的产物是一种高硫酸化的硫酸软骨素,具有很高的药用价值。与市面上鲨鱼硫酸软骨素(Shark-CS)相比,除物种多样性导致的差异外,其余结构基本一致,与稀碱-酶解-化学沉淀法从鲟鱼软骨中分离得到的硫酸软骨素相似,且回收率达93.68%。由此可知,本发明制备方法中鲟鱼硫酸软骨素的流失量较小。
对比例1
一种鲟鱼硫酸软骨素的制备方法,步骤如下:
(1)鲟鱼软骨分割:将新鲜鲟鱼软骨加入水中加热,煮至骨肉分离,取出,打碎至颗粒状得到鲟鱼软骨颗粒备用;
(2)热液化技术(HP)前处理:将步骤(1)获得的鲟鱼软骨颗粒放置于高压反应釜中,加热至温度130℃,保温2h得到热液化软骨液,该条件下获得热液化鱼骨液的亮度值为37.5;(3)酶解:将步骤(2)获得的热液化软骨液按料液比1:2加水稀释,加入复合中性蛋白酶进行酶解,酶解条件为:温度40℃、时间6h、酶添加量0.1%;待酶解完全后,于100℃、10min条件下灭酶得到酶解液,该条件下蛋白的水解度为17;
(4)超滤分离:将步骤(3)获得的酶解液依次通过陶瓷膜过滤、RO反渗透膜浓缩、10kDa卷式膜超滤分离得到鲟鱼硫酸软骨素溶液。
经检测,对比例1所得鲟鱼硫酸软骨素的回收率为73.89%。
对比例2
(1)鲟鱼软骨分割:将新鲜鲟鱼软骨加入水中加热,煮至骨肉分离,取出,打碎至颗粒状得到鲟鱼软骨颗粒备用;
(2)蒸汽爆破技术(SE)前处理:将步骤(1)获得的鲟鱼软骨颗粒放置于蒸汽爆破机中,在1Mpa条件下处理240s,该条件下获得蒸汽爆破软骨液的亮度值为78;
(3)酶解:将步骤(2)获得的蒸汽爆破软骨液经预冻后,放置于真空冷冻干燥箱中,以-80℃、真空度20Pa、48h条件冻干后,按料液比1:40加水稀释,加入复合中性蛋白酶进行酶解,酶解条件为:温度55℃、时间9h、酶添加量1.0%;待酶解完全后,于100℃、10min条件下灭酶得到酶解液,该条件下蛋白的水解度为21%;
(4)超滤分离:将步骤(3)获得的酶解液依次通过陶瓷膜过滤、RO反渗透膜浓缩、10kDa卷式膜超滤分离得到鲟鱼硫酸软骨素溶液。
经检测,对比例2所获得的鲟鱼软骨素平均分子量为62.0±0.18kDa,蛋白质含量为23.02±0.3%,糖醛酸含量为28.24±0.99%,硫酸根含量为12.1±0.3%,回收率为80.60%。
实施例1步骤(2)所得的鲟鱼软骨颗粒以及未经前处理的鲟鱼软骨颗粒的微观结构如图2所示,实施例1中热液化处理后的鲟鱼软骨颗粒效果最佳,呈现均匀碎片状,可有效地破坏鱼骨的结构,有利于后续酶解处理。对比例1的热液化条件与实施例1不同,其对鱼骨的破坏效果差于实施例1,最终得到的鲟鱼硫酸软骨素的回收率也低于实施例1。而对比例2表明蒸汽爆破的效果差于热液化。
实施例2
一种促糖尿病慢性伤口愈合敷料,由如下步骤制备得到:
1)将实施例1获得的鲟鱼硫酸软骨素溶液经冻干,加水质量比为3wt%复溶后,再加入氯化钙和羧甲基壳聚糖混合,鲟鱼硫酸软骨素、氯化钙和羧甲基壳聚糖的质量比为2:3:2,匀速搅拌,获得鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖复合物填料;
2)将羧甲基壳聚糖经溶解后-20℃低温冷冻,再-60℃超低温冷冻,最后冻干,重复6次,获得羧甲基壳聚糖海绵;
3)将羧甲基壳聚糖海绵与鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖复合物填料等比例于水溶液混合,水溶液中的质量含量为5wt%,再经溶解后-20℃低温冷冻,再-60℃超低温冷冻,最后冻干,重复5次,获得鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖/羧甲基壳聚糖复合海绵水凝胶敷料。
经细胞实验检测实施例2所得敷料对小鼠成纤维细胞(L929)细胞增殖及迁移活性的影响;并通过动物实验测试敷料对糖尿病小鼠慢性创口愈合性能,监测小鼠血清中炎症因子变化。结果表明,热液化鲟鱼硫酸软骨素敷料在浓度6.25到100μg/mL的范围内对L929细胞无毒性作用;鲟鱼硫酸软骨素敷料孵育细胞的增殖率与孵育时间呈正相关;采用细胞划痕试验,对L929细胞迁移的影响结果如图3所示;对巨噬细胞免疫调节活性的影响结果如图4所示,实施例2敷料处理后的细胞形态由圆形变为纺锤形,呈扁平状,这表明实施例2敷料具有激活巨噬细胞的作用;MTT法测试表明实施例2敷料对RAW264.7细胞是无毒的;小鼠伤口愈合实验结果如图5所示,实施例2敷料和阳性对照均具有促进伤口愈合的作用。对小鼠伤口皮肤组织进行免疫组化分析,评估CD31、VEGF和PCNA的表达,结果如图6所示,实施例2敷料治疗组及阳性组的CD31、VEGF、PCNA表达明显上调,且效果优于阳性对照组,这说明实施例2敷料对小鼠伤口有显著的促血管生成作用,细胞增殖生物标志物PCNA的上调说明实施例2敷料能促进细胞增殖,这与L929细胞增殖的结果一致。
上述检测结果证明本发明的促糖尿病慢性伤口愈合敷料具有良好的促伤口愈合效果,可以尽快止血,通过促进成纤维细胞增殖、迁移、激活巨噬细胞、上调CD31、VEGF和PCNA的表达促进表皮和真皮增生、血管再生,有效降低伤口的炎症反应,加速小鼠皮肤伤口愈合。本发明的促糖尿病慢性伤口愈合敷料具有良好的产业化应用前景。
Claims (10)
1.一种鲟鱼硫酸软骨素的制备方法,其特征是,包括如下步骤:
(1)将鲟鱼软骨颗粒热液化处理;
(2)向处理后的鲟鱼软骨颗粒加入水和蛋白酶进行酶解,待酶解完全后加热灭酶得到酶解液;
(3)将酶解液过滤纯化得到鲟鱼硫酸软骨素溶液。
2.根据权利要求1所述的一种鲟鱼硫酸软骨素的制备方法,其特征是,所述步骤(1)中鲟鱼软骨颗粒由将新鲜鲟鱼软骨煮至骨肉分离后打碎得到,或由干制鲟鱼软骨与水混合至固液比为1:(3~4)后打碎得到。
3.根据权利要求1所述的一种鲟鱼硫酸软骨素的制备方法,其特征是,所述步骤(1)的热液化处理为将鲟鱼软骨颗粒进行热液化,热液化条件为压力0.04~0.10MPa、温度105~115℃、时间1.5~2.5 h。
4.根据权利要求1所述的一种鲟鱼硫酸软骨素的制备方法,其特征是,所述步骤(2)中蛋白酶为复合中性蛋白酶,由质量配比为(1~2):(1~2):(2~3)的风味蛋白酶、中性蛋白酶和木瓜蛋白酶组成,蛋白酶的添加量为0.2~0.8%。
5.根据权利要求1或4所述的一种鲟鱼硫酸软骨素的制备方法,其特征是,所述步骤(3)中,处理后的鲟鱼软骨颗粒和水的料液比为1:(3~6),酶解时温度为45~60℃,酶解时间为6~9 h;加热至90~100℃后保温10~20min灭酶。
6.根据权利要求1所述的一种鲟鱼硫酸软骨素的制备方法,其特征是,所述步骤(4)中过滤纯化过程为将酶解液依次通过陶瓷膜过滤、RO反渗透膜浓缩、10 kDa卷式膜超滤分离。
7.一种促糖尿病慢性伤口愈合敷料的制备方法,其特征是,包括如下步骤:
1)将鲟鱼硫酸软骨素溶液经冻干、复溶后,再加入可溶性钙盐和羧甲基壳聚糖交联,得到鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖复合物填料;
2)将羧甲基壳聚糖冻融1~6次后,冻干得到羧甲基壳聚糖海绵;
3)将羧甲基壳聚糖海绵与填料混合得到鲟鱼硫酸软骨素-Ca-羧甲基壳聚糖/羧甲基壳聚糖复合海绵水凝胶敷料。
8.根据权利要求7所述的一种促糖尿病慢性伤口愈合敷料的制备方法,其特征是,所述步骤1)中鲟鱼硫酸软骨素和可溶性钙盐中钙离子的摩尔比为(1~2):(3~5),鲟鱼硫酸软骨素和羧甲基壳聚糖的质量比为(0.5~1): 1。
9.根据权利要求7所述的一种促糖尿病慢性伤口愈合敷料的制备方法,其特征是,所述步骤2)的冻融过程为将羧甲基壳聚糖溶解后-10~-20℃低温冷冻,再-30~-60℃超低温冷冻,最后冻干。
10.根据权利要求7所述的一种促糖尿病慢性伤口愈合敷料的制备方法,其特征是,所述步骤3)中羧甲基壳聚糖海绵与填料的质量比为1:(1~3)。
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