CN111647098A - 一种鱼皮来源的硫酸软骨素/硫酸皮肤素及其应用 - Google Patents
一种鱼皮来源的硫酸软骨素/硫酸皮肤素及其应用 Download PDFInfo
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- CN111647098A CN111647098A CN202010552297.8A CN202010552297A CN111647098A CN 111647098 A CN111647098 A CN 111647098A CN 202010552297 A CN202010552297 A CN 202010552297A CN 111647098 A CN111647098 A CN 111647098A
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- fish
- sulfate
- chondroitin sulfate
- fish skin
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Abstract
本发明涉及一种鱼皮来源的硫酸软骨素/硫酸皮肤素及其应用。本发明首次公开来自三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素,鱼皮种类不同,制备出的硫酸软骨素/硫酸皮肤素的结构及二糖组分也不同,龙利鱼鱼皮硫酸软骨素/硫酸皮肤素的主要二糖组成为iA‑unit,iO‑unit与A‑unit;绿鳍马面鲀鱼为iA‑unit,iO‑unit与iC‑unit;比目鱼为iA‑unit,A‑unit,iO‑unit与O‑unit。本发明中的三种鱼类的鱼皮多不被食用,作为废料丢弃,利用鱼皮制备硫酸软骨素/硫酸皮肤素,可以变废为宝,同时扩大硫酸软骨素/硫酸皮肤素的原料来源。
Description
技术领域
本发明涉及一种鱼皮来源的硫酸软骨素/硫酸皮肤素及其应用,属于生物技术技术领域。
背景技术
糖胺聚糖(Glycosaminoglycan,GAG)是由己糖醛酸和己糖胺组成的二糖单位重复连接而成的聚阴离子直链杂多糖,广泛存在于细胞表面以及细胞外基质中(Sugahara.2003,Miyata and Kitagawa.2017)。GAGs根据单糖残基、残基间糖苷键类型以及硫酸基数目和位置的不同,可以分为透明质酸(Hyaluronic acid,HA)、硫酸软骨素(Chondroitin sulfate,CS)/硫酸皮肤素(Dermatan sulfate,DS)、硫酸角质素(Keratansulfate,KS)和肝素(Heparin,Hep)/硫酸乙酰肝素(Heparan sulfate,HS)四大类。
CS/DS是一类主要的GAGs,CS由D-葡萄糖醛酸(D-glucuronic acid,GlcUA)和N-乙酰基-D-半乳糖胺(N-acetyl-D-galactosamine,GalNAc)组成的二糖单位重复连接而成,其中D-葡萄糖醛酸在C-5差向异构酶的作用下变为L-艾杜糖醛酸(L-iduronic acid,IdoUA)而变成了DS(Silbert,J.E.2002,Maccarana,M.2006)。因此CS与DS链经常以CS-DS共聚结构存在于哺乳动物组织中(Izumikawa,T.2004,Cheng,F.1994)。在生物合成过程中,CS/DS链通常会在GalNAc的C-4位和/或C-6位,和/或GlcUA/IdoUA的C-2位上被磺基转移酶进一步修饰,以形成独特的结构域。这些特定的结构域可以与生长因子、细胞因子等生物分子相互作用,参与众多的生理病理过程,如参与细胞间信号转导;促进神经细胞分化及神经元生长;参与癌症的发生发展等过程,因此在医学药学等领域具有广范的研究和应用价值(Sugahara,K.2003,Takuo Nakano.2010,Charalampos G.2014)。
研究表明不同种属动物体内含有的CS/DS种类、含量各不相同。海洋是一个巨大的生物资源库,海洋动物体内的GAGs在分子量与硫酸化度上与陆生生物有所不同。含有结构多样化的CS/DS在海洋中大量存在,广泛参与着细胞的增殖分化,促进损伤修复,参与细胞间信号转导等过程,因此开发新来源的海洋CS/DS有着重要的意义。目前海洋动物是CS/DS的重要来源,如富含C-unit的CS-C来源于鲨鱼软骨,富含D unit的CS-D来源于鲨鱼翅,富含E unit的CS-E来源于鱿鱼软骨。中国专利文献CN103554304A(申请号201310548309.X)公开了一种用鲟鱼脊骨制备低分子量鲟鱼硫酸软骨素的方法;中国专利文献CN104862366A(申请号201510340198.2)公开了一种从黑色鲨鱼皮提取胶原肽、硫酸皮肤素、羟基磷灰石和黑色素的工艺。鱼类是一类主要的海洋生物资源,含量丰富,鱼类的肉质鲜美,营养丰富,历来深受广大消费者喜爱,但很多鱼类的鱼皮因为口味较差,不宜食用等原因,在加工过程中往往被作为废料丢弃,造成环境污染与资源浪费。
发明内容
本发明针对现有技术的不足,提供了一种鱼皮来源的硫酸软骨素/硫酸皮肤素及其应用。
术语说明:
O-unit指不发生硫酸化的硫酸软骨素(CS)的二糖单位;
C-unit指N-乙酰基-D-半乳糖胺C-6位发生硫酸化的硫酸软骨素(CS)的二糖单位;
A-unit指N-乙酰基-D-半乳糖胺C-4位发生硫酸化的硫酸软骨素(CS)的二糖单位;
D-unit指N-乙酰基-D-半乳糖胺C-6位和葡萄糖醛酸C-2位发生硫酸化的硫酸软骨素(CS)的二糖单位;
B-unit指N-乙酰基-D-半乳糖胺C-4位和葡萄糖醛酸C-2位发生硫酸化的硫酸软骨素(CS)的二糖单位;
E-unit指N-乙酰基-D-半乳糖胺C-4位和C-6位发生硫酸化的硫酸软骨素(CS)的二糖单位;
iO-unit指不发生硫酸化的硫酸皮肤素(DS)的二糖单位;
iC-unit指N-乙酰基-D-半乳糖胺C-6位发生硫酸化的硫酸皮肤素(DS)的二糖单位;
iA-unit指N-乙酰基-D-半乳糖胺C-4位发生硫酸化的硫酸皮肤素(DS)的二糖单位;
iD-unit指N-乙酰基-D-半乳糖胺C-6位和艾杜糖醛酸C-2位发生硫酸化的硫酸皮肤素(DS)的二糖单位;
iB-unit指N-乙酰基-D-半乳糖胺C-4位和艾杜糖醛酸C-2位发生硫酸化的硫酸皮肤素(DS)的二糖单位;
iE-unit指N-乙酰基-D-半乳糖胺C-4位和C-6位发生硫酸化的硫酸皮肤素(DS)的二糖单位。
室温:具有本领域公知的含义,一般是指25±2℃。
本发明的技术方案如下:
一种鱼皮来源的硫酸软骨素/硫酸皮肤素,分子量为30-100KD,二糖组成如下,鱼皮选自龙利鱼,绿鳍马面鲀鱼,比目鱼的鱼皮中的一种:
二糖单位 | 质量百分比 |
O-unit | 4.32%-11.54% |
C-unit | 2.54%-10% |
A-unit | 5.07%-30.87% |
D-unit | 0.1%-1% |
B-unit | 0.01%-0.18% |
E-unit | 0.1%-1% |
iO-unit | 12.47%-20% |
iC-unit | 5.06%-13.93% |
iA-unit | 26.01%-56.14% |
iD-unit | 0.43%-2% |
iB-unit | 2.26%-5.58% |
iE-unit | 0.47%-3.29% |
根据本发明优选的,一种龙利鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为56.24±20KD,二糖组成如下:
二糖单位 | 质量百分比 |
O-unit | 5.42±1.1% |
C-unit | 3.32±0.66% |
A-unit | 11.56±0.12% |
D-unit | 0.21±0.11% |
B-unit | 0.08±0.04% |
E-unit | 0.32±0.06% |
iO-unit | 13.96±1.4% |
iC-unit | 8.10±1.62% |
iA-unit | 48.70±4.87% |
iD-unit | 1.09±0.22% |
iB-unit | 4.65±0.93% |
iE-unit | 2.59±0.52% |
根据本发明优选的,一种绿鳍马面鲀鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为6052±20KD,二糖组成如下:
二糖单位 | 质量百分比 |
O-unit | 5.75±1.2% |
C-unit | 3.18±0.64% |
A-unit | 6.34±1.27% |
D-unit | 0.25±0.13% |
B-unit | 0.03±0.02% |
E-unit | 0.46±0.23% |
iO-unit | 13.86±1.39% |
iC-unit | 12.66±1.27% |
iA-unit | 51.04±5.10% |
iD-unit | 0.86±0.43% |
iB-unit | 2.82±0.56% |
iE-unit | 2.74±0.55% |
根据本发明优选的,一种比目鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为65.19±20KD,二糖组成如下:
根据本发明优选的,所述鱼皮来源的硫酸软骨素/硫酸皮肤素的制备方法,包括如下步骤:
(1)将鱼皮剥离鱼体,去除鱼肉,清洗干净,剪碎后加入丙酮脱脂,得脱脂鱼皮;
(2)向步骤(1)制得的脱脂鱼皮中加入无水乙醇脱水,真空干燥,得脱脂脱水鱼皮;
(3)向步骤(2)制得的脱脂脱水鱼皮中加入三蒸水,煮沸后冷却至室温;再加入酶反应缓冲液,调节pH为5.0~9.0,加入脱脂脱水鱼皮干重1‰~5‰的蛋白酶进行酶解,固液分离取液体,制得酶解粗液;
(4)向步骤(3)制得的酶解粗液中加入终质量浓度2~8%的三氯乙酸,冰上沉淀,固液分离取液体,乙醚抽提,再按照质量体积比为1~10%加入醋酸钠粉末,单位为mg/mL;然后再加入1~5倍体积的无水乙醇,过夜沉淀,取沉淀,制得多糖粗品;
(5)将步骤(4)中制得的多糖粗品溶解于水,纯化,乙醇沉淀,固液分离取液体,超滤,干燥,制得鱼皮来源的硫酸软骨素/硫酸皮肤素。
根据本发明优选的,步骤(1)中所述丙酮脱脂是加入3~4倍体积的丙酮脱脂3~12h,重复3~5次。
根据本发明优选的,步骤(2)中所述无水乙醇脱水是加入3~4倍体积的无水乙醇脱水3~12h,重复3~5次。
根据本发明优选的,步骤(3)中所述脱脂脱水鱼皮与三蒸水的质量体积比为1:5,单位为mg/mL。
根据本发明优选的,步骤(3)中所述煮沸后的沸腾时间为30~50min;所述酶解温度为40~70℃,时间为24~96h。
根据本发明优选的,步骤(3)中所述酶反应缓冲液的组分:0.1M硼酸,10mM氯化钙,pH 8.0。
根据本发明优选的,步骤(3)中所述蛋白酶为黑曲霉蛋白酶、链霉蛋白酶E、木瓜蛋白酶、胰蛋白酶中的一种或多种。
根据本发明优选的,步骤(4)中所述冰上沉淀的时间为15~30min。
根据本发明优选的,步骤(4)中所述乙醚抽提是加入等体积的乙醚抽提3~4次。
根据本发明优选的,步骤(5)中所述纯化采用阴离子交换树脂纯化,0-2.5M氯化钠溶液浓度梯度洗脱。
上述鱼皮来源的硫酸软骨素/硫酸皮肤素在制备抗肿瘤药物和/或食品中的应用。
根据本发明优选的,所述在制备抗肿瘤药物的应用中硫酸软骨素/硫酸皮肤素可与合适的递送载体结合后给药;所述抗肿瘤药物通过口服或肠外给药。
上述鱼皮来源的硫酸软骨素/硫酸皮肤素在损伤修复中的应用。
根据本发明优选的,所述在损伤修复中的应用包括但不限于通过敷布、纱布、或涂抹方式经过皮肤途径渗透。
本发明中三种鱼类鱼皮来源的硫酸软骨素/硫酸皮肤素可以涂敷到合适的敷贴上(如4-5层纱布),敷贴应覆盖受损区域,且敷贴本身用绷带固定;应用的剂量取决于受损面积,用量为5-100g/cm2;敷贴应每天使用12-24小时,连续使用10-50天。
根据本发明优选的,所述硫酸软骨素/硫酸皮肤素在损伤修复中的剂型为溶液,悬浮液,凝胶剂,糊剂,软膏或乳膏,包含药物和化妆品领域可接受的稀释剂、辅助剂和/或赋形剂,其中包括缓冲剂如磷酸盐、柠檬酸盐、醋酸盐和其他有机酸盐;抗氧化剂如抗坏血酸;肽、蛋白质如血清白蛋白、明胶或免疫球蛋白;亲水性聚合物如聚乙烯吡咯烷酮、天然的或合成的油;氨基酸如甘氨酸、谷氨酸、天冬氨酸或精氨酸;单糖、二糖和其他碳水化合物,包括纤维素或其衍生物、葡萄糖、乳糖、甘露糖或糊精;螯合剂如EDTA;糖醇如甘露糖醇或山梨糖醇;无机盐,如氯化钠;非离子表面活性剂如吐温、聚乙二醇。
一种用于损伤修复的硫酸软骨素/硫酸皮肤素制剂,是将上述鱼类来源的源硫酸软骨素/硫酸皮肤素的冻干粉末悬浮或溶解于含有0.1-10wt%蔗糖、1-20wt%甘油的溶剂中,悬浮液或溶液的pH为6.5-8.5,溶剂为水。
有益效果:
1、本发明首次公开了来自三种鱼类(包括龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素,鱼皮种类不同,制备出的硫酸软骨素/硫酸皮肤素的结构及二糖组分也不同。本发明中的三种鱼类的鱼皮多不被食用,作为废料丢弃,利用龙利鱼、绿鳍马面鲀鱼、比目鱼的鱼皮制备硫酸软骨素/硫酸皮肤素,可以变废为宝,同时扩大硫酸软骨素/硫酸皮肤素的原料来源。
2、硫酸软软骨素/硫酸皮肤素因其结构特征,经常以CS-DS共聚结构存在于哺乳动物组织中。以往鱼类资源的利用报道通常仅关注于硫酸软软骨素或仅关注于硫酸皮肤素,而未将其共同分析。本发明首次将三种鱼类(包括龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软软骨素/硫酸皮肤素共同分析。
3、本发明制得的龙利鱼、绿鳍马面鲀鱼、比目鱼鱼皮来源的硫酸软骨素/硫酸皮肤素在结构及二糖组分上具有特异性,在抗肿瘤实验中具有抑制肿瘤细胞增殖活性的能力,在200μg/mL时抑制肿瘤细胞增殖活性可达60%,为研发新型抗肿瘤糖类功能食品和药物以及化妆品提供了原料和理论基础。
附图说明
图1为本发明制备的三种鱼类(A龙利鱼,B绿鳍马面鲀鱼,C比目鱼)鱼皮来源的硫酸软骨素/硫酸皮肤素经Hyaluronidase、Chondroteinase AC II、Chondroteinase B、Heparinase I II III生物酶降解后的糖胺聚糖种类组成HPLC分析图谱;图中,横坐标为保留时间,纵坐标为紫外强度;
图2为本发明制备的三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮来源的硫酸软骨素/硫酸皮肤素分子量检测图谱;图中,横坐标为保留时间,纵坐标为紫外强度;
图3为本发明制备的三种鱼类(A龙利鱼,B绿鳍马面鲀鱼,C比目鱼)鱼皮来源的硫酸软骨素/硫酸皮肤素经Chondroteinase AC II降解后的CS二糖组成图谱;图中,横坐标为保留时间,纵坐标为紫外强度;
图4为本发明制备的三种鱼类(A龙利鱼,B绿鳍马面鲀鱼,C比目鱼)鱼皮来源的硫酸软骨素/硫酸皮肤素经Chondroteinase B降解后的DS二糖组成图谱;图中,横坐标为保留时间,纵坐标为紫外强度;
图5为本发明制备的三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮来源的硫酸软骨素/硫酸皮肤素抗肿瘤细胞增殖活性统计分析折线图;图中,A为293T细胞,B为4T1细胞,C为67NR细胞,横坐标为硫酸软骨素/硫酸皮肤素浓度;纵坐标为相对细胞活性。
具体实施方案
下面结合实施例进一步全面公开本发明如何实施的技术方案,这些实施例不能理解为是为了限制本发明的应用范围。发明人已经尽最大努力确保实施例中各参数的准确性(例如量,温度,等等),但是一些实验误差和偏差也应该予以考虑。除非另有说明,本发明中分子量是指平均分子量,温度是指摄氏度。实施例中涉及的材料,若无特殊说明,均为普通市售产品。
原料来源:
龙利鱼(Sole fish),绿鳍马面鲀鱼(Navo donseptentrionalis),比目鱼(Flounder)均购置于山东青岛即墨海鲜批发市场。
实施例1
三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素的制备方法,包括如下步骤:
(1)将三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮分别剥离,去除鱼肉,清洗干净,称重并记录;将鱼皮用剪刀剪碎后放入锥形瓶中,加入3倍体积的丙酮脱脂3~12h,倒掉丙酮,重复脱脂3次,得脱脂鱼皮;
(2)向步骤(1)的脱脂鱼皮中加入3倍体积的无水乙醇脱水3~12h,倒掉无水乙醇,重复脱水3次;放入真空干燥箱中干燥,称重,得脱脂脱水鱼皮;
(3)将步骤(2)的脱脂脱水鱼皮放入锥形瓶中,按照质量体积比(mg/mL)为1:5加入三蒸水,加热煮沸,沸腾30min后冷却至室温;再加入酶反应缓冲液(0.1M硼酸,10mM氯化钙,pH 8.0),将样品溶液pH调节至8.0,加入脱脂脱水鱼皮干重1‰的黑曲霉蛋白酶,50℃反应72h,每24h补加酶液一次,之后煮沸10min将酶灭活,固液分离取液体,制得酶解粗液;
(4)向步骤(3)制得的酶解粗液中加入终质量浓度为5%的三氯乙酸(TCA),冰上沉淀15min,10000rpm离心15min,弃掉沉淀取上清,加入等体积乙醚抽提3次;再按照质量体积比(mg/mL)为5%加入醋酸钠粉末,并加入3倍体积的无水乙醇,过夜沉淀;10000rpm离心收集沉淀,将沉淀在真空干燥箱中干燥制得多糖粗品;
(5)将步骤(4)中制得的多糖粗品溶于水,浓度为10mg/mL,加入多糖粗品重量60%的A98阴离子交换树脂(购于德国朗盛公司),室温孵育1h后用0.5M,1M,2M氯化钠溶液梯度洗脱,2M氯化钠洗脱液用乙醇沉淀,并用10KDa孔径超滤膜超滤脱盐,干燥后分别制得三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素。
实施例2、三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素中糖胺聚糖种类及其含量的分析
三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素分别用Hyaluronidase、Chondroteinase AC II、Chondroteinase B、Heparinase I II III生物酶在各自最适反应条件下处理,然后用HPLC分析,分析条件为凝胶柱:SuperdexTM peptide10/300GL;流动相:0.2M NH4HCO3;流速:0.4mL/min;检测器:岛津紫外检测器(SPD-20A)。
其中Hyaluronidase专一性降解透明质酸(HA),Chondroteinase AC II专一性降解硫酸软骨素(CS),Chondroteinase B专一性降解硫酸皮肤素(DS),Heparinase I II III专一性降解肝素(Hep)和硫酸乙酰肝素(HS)。
检测结果如图1所示,三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素仅在Chondroteinase AC II和Chondroteinase B降解酶的作用下出现降解产物(如箭头所示),其他酶作用下无降解产物出现。因此三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素中几乎不含HA和Hep/HS,主要是CS和DS。
实施例3、三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素分子量分析
将平均分子量为12KD、50KD、150KD、270KD、410KD的商品化葡聚糖和三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素配置成10mg/mL的溶液,均取100μL进行HPLC检测分析。
检测条件为:色谱柱:UltrahydrogelTM100 7.8×300mm(Waters);流动相:0.02MNaH2PO4,0.02M Na2HPO4,0.02%NaN3;流速:0.6mL/min;检测器:岛津示差检测器(RID-10A)。
检测结果如图2所示,以各分子量葡聚糖标准的出峰时间为横坐标,以各葡聚糖的分子量的常用对数lgMr为纵坐标,作线性回归分析,绘制标准曲线,通过标准曲线得到的线性回归方程:Y=-2.7284X+26.624,R2=0.9948,由此计算出三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素的平均分子量:龙利鱼的平均分子量为56.24±20KD,绿鳍马面鲀鱼的平均分子量为60.52±20KD,比目鱼的平均分子量为65.19±20KD。
实施例4、三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素二糖组成含量分析
(1)用商品化的Chondroteinase AC II(专一性降解硫酸软骨素)和Chondroteinase B(专一性降解硫酸皮肤素)分别对质量浓度为1%的三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素溶液,在各自的最适反应条件下分别降解2h,反应产物用离心浓缩仪干燥;
(2)反应产物用2-氨基苯甲酰胺标记:按14:6:1:1的质量比例混合二甲基亚砜,冰醋酸,氰基硼氢化钠和2-氨基苯甲酰胺,混合后取5μL加入到步骤(1)干燥后的反应产物中,65℃反应2h,反应产物用三氯甲烷抽提12次,之后用离心浓缩仪干燥;
(3)干燥产物加100μL三蒸水溶解,HPLC分析检测,检测条件为:色谱柱:YMC PackPA-G column(YMC,日本);流动相(梯度洗脱):60min内NaH2PO4的浓度从16mM线性升至460mM;流速:1mL/min;检测器:岛津荧光检测器(Ex330nm,Em420nm)。
检测结果如图3,图4所示,其中图3为三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素经Chondroteinase AC II降解后的CS二糖组成图谱;图4为三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素经ChondroteinaseB降解后的DS二糖组成图谱。通过峰面积积分得出各二糖组成成分质量百分比如表1~3。
表1.龙利鱼鱼皮的硫酸软骨素/硫酸皮肤素的二糖组成
二糖单位 | 质量百分比 |
O-unit | 5.42±1.1% |
C-unit | 3.32±0.66% |
A-unit | 11.56±0.12% |
D-unit | 0.21±0.11% |
B-unit | 0.08±0.04% |
E-unit | 0.32±0.06% |
iO-unit | 13.96±1.4% |
iC-unit | 8.10±1.62% |
iA-unit | 48.70±4.87% |
iD-unit | 1.09±0.22% |
iB-unit | 4.65±0.93% |
iE-unit | 2.59±0.52% |
表2.绿鳍马面鲀鱼鱼皮的硫酸软骨素/硫酸皮肤素的二糖组成
表3.比目鱼鱼皮的硫酸软骨素/硫酸皮肤素的二糖组成
二糖单位 | 质量百分比 |
O-unit | 10.49±1.05% |
C-unit | 6.55±1.31% |
A-unit | 28.06±2.81% |
D-unit | 0.50±0.25% |
B-unit | 0.12±0.06% |
E-unit | 0.20±0.1% |
iO-unit | 13.90±1.39% |
iC-unit | 6.32±1.26% |
iA-unit | 28.90±2.89% |
iD-unit | 0.86±0.43% |
iB-unit | 3.16±0.63% |
iE-unit | 0.95±0.48% |
本发明中三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素在结构上与已知的其他鱼类鱼皮在二糖组成上有所不同。如已知鮟鱇鱼鱼皮的主要二糖组成为iA-unit(张虹,中国食品学报,2008)。而本发明中,龙利鱼鱼皮硫酸软骨素/硫酸皮肤素的主要二糖组成为iA-unit,iO-unit与A-unit;绿鳍马面鲀鱼鱼皮硫酸软骨素/硫酸皮肤素的主要二糖组成为iA-unit,iO-unit与iC-unit;比目鱼鱼皮硫酸软骨素/硫酸皮肤素的主要二糖组成为iA-unit,A-unit,iO-unit与O-unit。这也表明了不同种属动物体内含有的CS/DS种类、含量各不相同。
实施例5、三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素体外抗肿瘤活性检测
培养人肾上皮细胞株293T细胞和乳腺癌细胞株4T1细胞与67NR细胞,培养条件均为:在含2mM非必须氨基酸、2mM L-谷氨酰胺的DMEM培养液中加入10%胎牛血清,100U/mL青霉素、100μg/mL链霉素双抗加入合适浓度的细胞,37℃,5%CO2培养箱中培养。
硫酸软骨素/硫酸皮肤素体外抗肿瘤活性检测步骤如下:
(1)在96孔细胞培养板中分别加入5000个293T细胞,4T1细胞与67NR细胞培养24h;
(2)在三种细胞中分别加入不同终浓度(0、25、50、100、200μg/mL)的三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素,继续培养24h;
(3)24h后,在培养液中加入200μL的MTT溶液(5mg/mL),继续培养4h;
(4)4h后,小心吸去孔内培养液,每孔加入150μL二甲基亚砜,摇床低速震荡10min使结晶物充分溶解,490nm下检测吸光值。
检测结果如图5所示,加入三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素后的293T细胞数目影响不大,但加入三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素的4T1细胞和67NR细胞的数目有所降低;且随着多糖浓度的增加,抗肿瘤细胞增殖的能力逐渐增强,在200μg/mL多糖的作用下,细胞数目仅达到无糖作用时的40-60%。以上结果表明龙利鱼,绿鳍马面鲀鱼,比目鱼鱼皮的硫酸软骨素/硫酸皮肤素均不具有较高的细胞毒性,对正常细胞的增殖影响不大,但均具有抑制肿瘤细胞增殖的能力,可为研发新型抗肿瘤糖类药物和/或食品提供原料和理论基础。
本发明中三种鱼类(龙利鱼,绿鳍马面鲀鱼,比目鱼)鱼皮的硫酸软骨素/硫酸皮肤素具有明确的抑制肿瘤细胞增殖活性的能力,在200μg/mL时抑制肿瘤细胞增殖活性可达60%,可为研发新型抗肿瘤糖类药物和/或食品提供原料和理论基础。与此相比,已知的鮟鱇鱼鱼皮硫酸皮肤素并未明确表明其可抑制肿瘤细胞增殖的活性。
Claims (10)
1.一种鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,分子量为30-100KD,二糖组成如下,鱼皮选自龙利鱼,绿鳍马面鲀鱼,比目鱼的鱼皮中的一种:
。
2.如权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,龙利鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为56.24±20KD,二糖组成如下:
。
3.如权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,绿鳍马面鲀鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为60.52±20KD,二糖组成如下:
。
4.如权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,比目鱼鱼皮的硫酸软骨素/硫酸皮肤素,分子量为65.19±20KD,二糖组成如下:
。
5.如权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,其制备方法,包括如下步骤:
(1)将鱼皮剥离鱼体,去除鱼肉,清洗干净,剪碎后加入丙酮脱脂,得脱脂鱼皮;
(2)向步骤(1)制得的脱脂鱼皮中加入无水乙醇脱水,真空干燥,得脱脂脱水鱼皮;
(3)向步骤(2)制得的脱脂脱水鱼皮中加入三蒸水,煮沸后冷却至室温;再加入酶反应缓冲液,调节pH为5.0~9.0,加入脱脂脱水鱼皮干重1‰~5‰的蛋白酶进行酶解,固液分离取液体,制得酶解粗液;
(4)向步骤(3)制得的酶解粗液中加入终质量浓度2~8%的三氯乙酸,冰上沉淀,固液分离取液体,乙醚抽提,再按照质量体积比为1~10%加入醋酸钠粉末,单位为mg/mL;然后再加入1~5倍体积的无水乙醇,过夜沉淀,取沉淀,制得多糖粗品;
(5)将步骤(4)中制得的多糖粗品溶解于水,纯化,乙醇沉淀,固液分离取液体,超滤,干燥,制得鱼皮来源的硫酸软骨素/硫酸皮肤素。
6.如权利要求5所述的鱼皮来源的硫酸软骨素/硫酸皮肤素,其特征在于,满足以下条件之一项或多项:
i.步骤(1)中所述丙酮脱脂是加入3~4倍体积的丙酮脱脂3~12h,重复3~5次;
ii.步骤(2)中所述无水乙醇脱水是加入3~4倍体积的无水乙醇脱水3~12h,重复3~5次;
iii.步骤(3)中所述脱脂脱水鱼皮与三蒸水的质量体积比为1:5,单位为mg/mL;
iv.步骤(3)中所述煮沸后的沸腾时间为30~50min;所述酶解温度为40~70℃,时间为24~96h;
v.步骤(3)中所述酶反应缓冲液的组分:0.1M硼酸,10mM氯化钙,pH 8.0;
vi.步骤(3)中所述蛋白酶为黑曲霉蛋白酶、链霉蛋白酶E、木瓜蛋白酶、胰蛋白酶中的一种或多种;
vii.步骤(4)中所述冰上沉淀的时间为15~30min;
viii.步骤(4)中所述乙醚抽提是加入等体积的乙醚抽提3~4次;
ix.步骤(5)中所述纯化采用阴离子交换树脂纯化,0-2.5M氯化钠溶液浓度梯度洗脱。
7.权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素在制备抗肿瘤药物和/或食品中的应用。
8.权利要求1所述的鱼皮来源的硫酸软骨素/硫酸皮肤素在损伤修复中的应用。
9.如权利要求8所述的在损伤修复中的应用,其特征在于,所述应用包括通过敷布、纱布、或涂抹方式经过皮肤途径渗透。
10.一种用于损伤修复的硫酸软骨素/硫酸皮肤素制剂,其特征在于,是将权利要求1所述的鱼类来源的源硫酸软骨素/硫酸皮肤的冻干粉末悬浮或溶解于含有0.1-10wt%蔗糖、1-20wt%甘油的溶剂中,悬浮液或溶液的pH为6.5-8.5,溶剂为水。
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