CN115433749A - 一种新型多岩藻糖支链fcs、制备方法及其缓解高糖损伤的应用 - Google Patents
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Abstract
本发明提供一种新型多岩藻糖支链FCS、制备方法及其缓解高糖损伤的应用,将海参粉胶原酶和基质金属蛋白酶初步酶解、光谱蛋白酶继续酶解,经醇沉得粗多糖后,一步膜分离即可得纯净FCS,经核磁结构测定和三级质谱测定主链二糖结构,得到FCS的精细结构为含双岩藻糖结构的FCS多糖,FCS可缓解高糖浸泡对于斑马鱼胚胎的损伤。
Description
技术领域
本发明涉及医疗保健技术领域,具体地说是一种新型多岩藻糖支链FCS、制备方法及其缓解高糖损伤的应用。
背景技术
众所周知,糖尿病是一种常见的代谢性疾病,胰岛素缺陷或胰岛素抵抗导致的糖相关代谢异常是糖尿病的主要发病机制。目前世界上DM患病人数约4.2亿,预计2040年患病人数将增至6.42亿。根据发病原因,糖尿病可被细分为Ⅰ型,Ⅱ型,妊娠和其他类型。其中,I型糖尿病以胰岛β细胞功能异常为发病机理,表现为胰岛素分泌不足,典型的治疗方法为胰岛素注射。而Ⅱ型糖尿病在总患病人数中的比例最大,约占成人糖尿病发病类型的90 %以上,且病症最复杂,常伴有肾脏损伤,心血管系统疾病,视网膜病变及泌尿系统损伤等与细胞损伤相关的并发症。长期高糖会对多种调控通路产生损伤,如产生氧化应激,而氧化应激是多种糖尿病并发症的基石。因此,寻找可减轻高糖损伤的天然药物,对II型糖尿病及其并发症的治疗具有重要意义。
海参是归属于无脊椎动物棘皮动物门(Echinodermata)海参纲(Holothuridea)的无脊椎动物,是海洋中重要的食物和药物资源,有着极高的营养价值,味道鲜美。近几年来,国内外在海参的生物活性物质及其药理作用等方面的研究较多,已经先后从几十种海参中分离获得了多种活性物质,包括多肽、蛋白质、多糖、脂类、三萜皂苷、凝集素、神经肽和糖肽。海参多糖目前研究较多的是海参硫酸软骨素 (FCS)等含有硫酸根的多糖,且海参硫酸酯多糖因其结构与肝素类似,目前对FCS抗糖尿病的研究多集中于对胰岛素抵抗等影响血糖指标的直接作用,而对于高糖损伤的应用研究较少。目前海参多糖的提取方法多为木瓜蛋白酶解法,但其实木瓜蛋白酶虽然为广谱蛋白酶,但是对于胶原蛋白的酶解能力并不强,因此需要的酶解时间过长,且其酶解温度较高,因此限制了其制备工艺的应用。已经有文献报道,海参FCS通过共价键连接于胶原蛋白纤维,因此需要找到更加特异性的酶,预先处理海参体壁,从而缩短制备时间,并提高得率。目前所得FCS结构皆为单岩藻糖支链,且主链的二糖结构很难确定。
发明内容
本发明的目的是解决上述现有技术的不足,提供一种新型多岩藻糖支链FCS、制备方法及其缓解高糖损伤的应用。
本发明解决其技术问题所采用的技术方案是:
一种新型多岩藻糖支链FCS,其特征在于多岩藻糖支链FCS的结构如下:
一种新型多岩藻糖支链FCS的制备方法,其特征在于制备方法的步骤如下:
(1)淡干海参粉碎,加入胶原酶和基质金属蛋白酶酶解;加入商品化广谱蛋白酶继续酶解,煮沸灭酶后,离心收集上清,醇沉后收集沉淀,重溶,透析得粗多糖溶液;
(2)粗多糖溶液通过截留分子量80-150 kDa膜一步分离,滤出液冻干即为纯FCS;
本发明所述的步骤(1)中所述胶原蛋白酶和基质金属蛋白酶的总添加量为2000U/g海参粉,酶解温度为 25~35 ℃,酶解时间为1-2 h;广谱蛋白酶选用菠萝蛋白酶、中性蛋白酶、木瓜蛋白酶中的任意一种或两种以上结合,酶解温度为25~35 ℃,酶解时间为3-5 h。
本发明所述的步骤(2)得到的纯FCS进行核磁测定,三级质谱法测定主链二糖结构。
本发明所述的三级质谱的样品前处理采用稀硫酸去除岩藻糖支链,硫酸软骨素酶ABC酶解得主链二糖;质谱参数为选用Obitrap系列质谱仪的数据依赖性负离子扫描模式,每一级扫描设置上一级扫描的响应值前三的离子片段触发离子打碎。扫描范围100~500Da,分辨率70000,锥孔电压100 V,碰撞能35 eV。
本发明所述的步骤(2)得到的纯FCS建立斑马鱼高糖损伤模型,测定FCS的治疗作用。
本发明所述的斑马鱼高糖损伤模型是选用斑马鱼胚胎,用葡萄糖浸泡法建立高糖损伤模型,葡萄糖浓度为100~200 mM,浸泡时间为2~3 d。
一种新型多岩藻糖支链FCS缓解高糖损伤的应用,其特征在于将多岩藻糖支链FCS用于缓解高糖损伤的机体中。
本发明由于采用上述方案,得到的多岩藻糖支链FCS可缓解高糖损伤。
说明书附图
图1为多糖对于斑马鱼胚胎的急性毒性实验结果图,显示在10-150 μg/mL浓度范围内,斑马鱼胚胎的存活率。
图2 为多糖缓解斑马鱼胚胎高糖损伤荧光显微镜图片,a图表示为对照表示未处理组,b图表示模型150 mM葡萄糖处理组,c图表示FCS-25 μg/mL,d图表示150 mM葡萄糖处理同时添加50 μg/mL多糖的药物处理组。
具体实施方式
下面对本发明进一步说明:
实施例1:
一种新型多岩藻糖支链FCS,多岩藻糖支链FCS的结构如下:
制备方法的步骤如下:
(1) 取淡干海参100 g,粉碎后,加入500 mL水,加入总酶活2 × 105 U任意配比的胶原酶和基质金属蛋白酶,25 ℃酶解1 h,加入1 g菠萝蛋白酶,30 ℃酶解5 h,煮沸灭酶,离心,上清浓缩后,加入4倍体积乙醇醇沉收集沉淀,加水重溶并透析,得粗多糖溶液;
(2) 粗多糖溶液通过截留分子量80 kDa膜一步分离,滤出液冻干即为纯FCS;
(3) 核磁测定FCS结构,FCS用0.1 mol/L硫酸于80 ℃水解20 min去除岩藻糖支链,加入0.1 U/mL硫酸软骨素ABC酶酶解过夜得主链二糖用于质谱分析;质谱参数为选用Obitrap系列质谱仪的数据依赖性负离子扫描模式,每一级扫描设置上一级扫描的响应值前三的离子片段触发离子打碎。扫描范围100~500 Da,分辨率70000,锥孔电压100 V,碰撞能35 eV。
(4) 选用受精1 d的斑马鱼胚胎,加入150 mM葡萄糖浸泡2 d,同时加入不同浓度FCS共浸泡,实验结束用荧光显微镜测定斑马鱼氧化应激水平。
实施例2:
一种新型多岩藻糖支链FCS,多岩藻糖支链FCS的结构如下:
制备方法的步骤如下::
(1) 取淡干海参100 g,粉碎后,加入500 mL水,加入总酶活2 × 105 U任意配比的胶原酶和基质金属蛋白酶,35 ℃酶解2 h,加入1 g木瓜蛋白酶,35 ℃酶解3 h,煮沸灭酶,离心,上清浓缩后,加入4倍体积乙醇醇沉收集沉淀,加水重溶并透析,得粗多糖溶液;
(2) 粗多糖溶液通过截留分子量100 kDa膜一步分离,滤出液冻干即为纯FCS;
(3) 核磁测定FCS结构,FCS用0.1 mol/L硫酸于80 ℃水解20 min去除岩藻糖支链,加入0.1 U/mL硫酸软骨素ABC酶酶解过夜得主链二糖用于质谱分析;质谱参数为选用Obitrap系列质谱仪的数据依赖性负离子扫描模式,每一级扫描设置上一级扫描的响应值前三的离子片段触发离子打碎。扫描范围100~500 Da,分辨率70000,锥孔电压100 V,碰撞能35 eV。
(4) 选用受精1 d的斑马鱼胚胎,加入100 mM葡萄糖浸泡3 d,同时加入不同浓度FCS共浸泡,实验结束用荧光显微镜测定斑马鱼氧化应激水平。
实施例3:
一种新型多岩藻糖支链FCS,多岩藻糖支链FCS的结构如下:
制备方法的步骤如下:
(1) 取淡干海参100 g,粉碎后,加入500 mL水,加入总酶活2 × 105 U任意配比的胶原酶和基质金属蛋白酶,30 ℃酶解1 h,加入1 g中性蛋白酶,25 ℃酶解5 h,煮沸灭酶,离心,上清浓缩后,加入4倍体积乙醇醇沉收集沉淀,加水重溶并透析,得粗多糖溶液;
(2) 粗多糖溶液通过截留分子量150 kDa膜一步分离,滤出液冻干即为纯FCS;
(3) 核磁测定FCS结构,FCS用0.1 mol/L硫酸于80 ℃水解20 min去除岩藻糖支链,加入0.1 U/mL硫酸软骨素ABC酶酶解过夜得主链二糖用于质谱分析;质谱参数为选用Obitrap系列质谱仪的数据依赖性负离子扫描模式,每一级扫描设置上一级扫描的响应值前三的离子片段触发离子打碎。扫描范围100~500 Da,分辨率70000,锥孔电压100 V,碰撞能35 eV。
(4) 选用受精1 d的斑马鱼胚胎,加入200 mM葡萄糖浸泡2 d,同时加入不同浓度FCS共浸泡,实验结束用荧光显微镜测定斑马鱼氧化应激水平。
实施例1-3与传统木瓜蛋白酶酶解法比较如下表1所示:
从上表可以看出,实施例1-3与传统木瓜蛋白酶酶解法相比较,酶解温度低、酶解时间短,多糖得率高,蛋白含量低,因此本发明提供的方法优于现有方法,在多糖的得率和纯度方面都比现行方法高效,其中,分别也对广谱蛋白酶选用菠萝蛋白酶、中性蛋白酶、木瓜蛋白酶至少任一两个组合或三个组成作为分析,其结果与实施例1-3的结果相同,都是酶解温度低、酶解时间短,多糖得率高,蛋白含量低。
图1为不同浓度FCS对于斑马鱼胚胎的急性毒性实验结果图,作为后续活性浓度的筛选,在10-50 μg/mL浓度,斑马鱼胚胎存活率为100%,FCS对于其无毒性,而从65 μg/mL开始,斑马鱼胚胎出现个体死亡,直至150 μg/mL,其死亡率为100%。
实施例1-3中分别得到相同的FCS,在进行斑马鱼高糖损伤模型时,采用的100 mM、150 mM、200 mM的葡萄糖进行高糖损伤,得到的多糖缓解斑马鱼胚胎高糖损伤荧光显微镜图片中都出现了同样的高糖损伤现象,附图2中b为150 mM的高糖损伤情况图,然后分别对150 mM的高糖损伤的斑马鱼进行添加FCS-25 μg/mL多糖的药物处理组和添加50 μg/mL多糖的药物处理组,分别对应图2中的c和d。
图2所示为多糖缓解斑马鱼胚胎高糖损伤,图中所选浓度为毒性筛选中无毒浓度中的最大两个浓度作为示例浓度,分别为25 μg/mL 和50 μg/mL ,a图表示为对照表示未处理组,b图表示模型150 mM葡萄糖处理组,c图表示150 mM葡萄糖处理同时添加FCS-25 μg/mL多糖的药物处理组,d图表示150 mM葡萄糖处理同时添加50 μg/mL多糖的药物处理组,注:白度越高,氧化应激程度越高,高糖损伤程度越高,从图中可以看出,本发明中所得FCS具有显著的降低高糖诱导的斑马鱼氧化应激水平的作用。
基质金属蛋白酶和胶原蛋白酶可以先将胶原蛋白四级结构解聚,便于后面进行广谱蛋白酶解,本发明证明(表1),利用这两种酶初步酶解,可以显著提高多糖的得率和纯度,优于目前常见的酶解方法。
Claims (7)
1.一种新型多岩藻糖支链FCS,其特征在于多岩藻糖支链FCS的结构如下:
一种新型多岩藻糖支链FCS的制备方法,其特征在于制备方法的步骤如下:
(1)淡干海参粉碎,加入胶原酶和基质金属蛋白酶酶解;加入商品化广谱蛋白酶继续酶解,煮沸灭酶后,离心收集上清,醇沉后收集沉淀,重溶,透析得粗多糖溶液;
(2)粗多糖溶液通过截留分子量80-150 kDa膜一步分离,滤出液冻干即为纯FCS;
根据权利要求2所述的一种新型多岩藻糖支链FCS的制备方法,其特征在于所述的步骤(1)中所述胶原蛋白酶和基质金属蛋白酶的总添加量为2000 U/g海参粉,酶解温度为 25~35 ℃,酶解时间为1-2 h;广谱蛋白酶选用菠萝蛋白酶、中性蛋白酶、木瓜蛋白酶中的任意一种或两种以上结合,酶解温度为25~35 ℃,酶解时间为3-5 h。
2.根据权利要求2所述的一种新型多岩藻糖支链FCS的制备方法,其特征在于所述的步骤(2)得到的纯FCS进行核磁测定,三级质谱法测定主链二糖结构。
3.根据权利要求4所述的一种新型多岩藻糖支链FCS的制备方法,其特征在于所述的三级质谱的样品前处理采用稀硫酸去除岩藻糖支链,硫酸软骨素酶ABC酶解得主链二糖;质谱参数为选用Obitrap系列质谱仪的数据依赖性负离子扫描模式,每一级扫描设置上一级扫描的响应值前三的离子片段触发离子打碎。
4.扫描范围100~500 Da,分辨率70000,锥孔电压100 V,碰撞能35 eV。
5.根据权利要求2所述的一种新型多岩藻糖支链FCS的制备方法,其特征在于所述的步骤(2)得到的纯FCS建立斑马鱼高糖损伤模型,测定FCS的治疗作用。
6.根据权利要求6所述的一种新型多岩藻糖支链FCS的制备方法,其特征在于所述的斑马鱼高糖损伤模型是选用斑马鱼胚胎,用葡萄糖浸泡法建立高糖损伤模型,葡萄糖浓度为100~200 mM,浸泡时间为2~3 d。
7.一种新型多岩藻糖支链FCS缓解高糖损伤的应用,其特征在于将多岩藻糖支链FCS用于缓解高糖损伤的机体中。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105695545A (zh) * | 2016-03-02 | 2016-06-22 | 集美大学 | 一种海参岩藻聚糖硫酸酯和海参糖蛋白的制备方法 |
KR20160149757A (ko) * | 2015-06-19 | 2016-12-28 | (주)뷰티화장품 | 효소-초고압 추출법을 이용한 해삼 추출물의 제조방법 및 이에 의해 제조된 해삼 추출물 |
WO2022067774A1 (zh) * | 2020-09-30 | 2022-04-07 | 牡丹江友搏药业有限责任公司 | 一种海参多糖的制备方法与应用 |
CN114316078A (zh) * | 2020-09-30 | 2022-04-12 | 牡丹江友搏药业有限责任公司 | 一种海参多糖的制备方法与应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20160149757A (ko) * | 2015-06-19 | 2016-12-28 | (주)뷰티화장품 | 효소-초고압 추출법을 이용한 해삼 추출물의 제조방법 및 이에 의해 제조된 해삼 추출물 |
CN105695545A (zh) * | 2016-03-02 | 2016-06-22 | 集美大学 | 一种海参岩藻聚糖硫酸酯和海参糖蛋白的制备方法 |
WO2022067774A1 (zh) * | 2020-09-30 | 2022-04-07 | 牡丹江友搏药业有限责任公司 | 一种海参多糖的制备方法与应用 |
CN114316078A (zh) * | 2020-09-30 | 2022-04-12 | 牡丹江友搏药业有限责任公司 | 一种海参多糖的制备方法与应用 |
Non-Patent Citations (1)
Title |
---|
P.-X. GONG ET AL.: "Structural elucidation and antidiabetic activity of fucosylated chondroitin sulfate from sea cucumber Stichopus japonicas", CARBOHYDRATE POLYMERS, vol. 262, 20 March 2021 (2021-03-20), pages 1 - 9 * |
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