CN110669152B - 一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法 - Google Patents
一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法 Download PDFInfo
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- CN110669152B CN110669152B CN201910995448.4A CN201910995448A CN110669152B CN 110669152 B CN110669152 B CN 110669152B CN 201910995448 A CN201910995448 A CN 201910995448A CN 110669152 B CN110669152 B CN 110669152B
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- oviductus ranae
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Abstract
本发明提供一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法。所述方法先将蛤蟆油充分溶胀后加入的胰蛋白酶酶解蛤蟆油，再通过DEAE阴离子交换色谱柱提取蛤蟆油中的糖胺聚糖OR‑GAG，然后经透析、冻干、浓缩后，用硫酸软骨素ABC酶特异性酶解OR‑GAG，足量的酶可将长链酶解至硫酸软骨素/硫酸皮肤素/透明质酸(CS/DS/HA)二糖单元。CS/DS/HA二糖单元经2‑氨基吖啶酮荧光标记后，利用LC/MS‑ITTOF进行结构分析，得到蛤蟆油中各二糖组分的结构与摩尔百分比。
Description
技术领域
本发明属于天然产物制备纯化领域，具体涉及一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法。
背景技术
蛤蟆油，又名雪蛤油、林蛙油、哈什蚂油，为雌蛙输卵管干制品。1g蛤蟆油中约有0.5 g粗蛋白、0.3 g无氮浸出物、0.1 g灰分、0.05 g脂肪以及0.02 g水分。蛤蟆油在《本草图经》和《本草纲目》中均有记载，且在明清时期，医学专家更是推崇蛤蟆油，并将其作为上等贡品敬献宫廷，其营养成分不亚于人参、燕窝等补品。蛤蟆油的传统功效有补肾益精、养阴润肺，主要用于肾虚精髓不足，眩晕耳鸣，病后或产后体虚气弱。现代医学又发现，其具有增强机体免疫力、提高抗氧化能力、降血脂、抗癌辅助作用等。其中，抗氧化、抗癌等功效可能与其含有硫酸软骨素/硫酸皮肤素/透明质酸有关。
硫酸软骨素(CS)、硫酸皮肤素(DS)以及透明质酸(HA)均属于糖胺聚糖(GAG),其中CS/DS是以乙酰氨基半乳糖(GalNAc)和己糖醛酸(GlcA/IdoA)为二糖单元构成的聚阴离子长链，而HA是由乙酰氨基葡萄糖(GlcNAc)和葡萄糖醛酸(GlcA)为二糖单元形成的唯一一种无硫酸化修饰的糖胺聚糖。CS/DS的修饰位点主要有GlcA的2位氧硫酸化，GalNAc的4位氧硫酸化和6位氧硫酸化，不同程度以及不同位点的硫酸化修饰致使其生物功能也有所不同。CS/DS的生物功能主要有促进软骨再生、抗凝血、抗血栓形成、抗衰老以及提高机体免疫力等，HA由于其长链上有大量羟基，具有很强的亲水作用，所以一般用于延缓皮肤衰老、润泽皮肤等保湿型化妆品中。
由于国内外未见有关蛤蟆油中CS/DS/HA的提取与结构分析的相关报道， 本发明开创性的建立了一种提取蛤蟆油中CS/DS/HA的方法，并首次对蛤蟆油中的CS/DS/HA进行结构解析，为深入研究蛤蟆油中CS/DS/HA的结构及其功效提供了原创性的提取途径和结构分析手段。
发明内容
本发明的目的在于提供一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法。该方法以蛤蟆油为原料，先用胰蛋白酶特异性酶解蛤蟆油蛋白，再利用阴离子交换色谱法将聚阴离子长链GAG分离出来，GAG中的NaCl通过透析进行除去、再冻干浓缩至无水状态，加入硫酸软骨素ABC酶特异性酶解CS/DS/HA长链至二糖单元，并通过分子排阻色谱法将得到的CS/DS/HA二糖单元进行纯化与分离。最后，CS/DS/HA二糖单元用荧光物质AMAC（2-氨基吖啶酮）进行标记，利用LC/MS-ITTOF对CS/DS/HA二糖单元进行结构分析，得到CS/DS/HA二糖的组分含量和摩尔百分比。
为实现上述目的，本发明采用如下技术方案：
一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法，包括以下步骤：
（1）胰蛋白酶酶解蛤蟆油：蛤蟆油经充分浸泡后用胰蛋白酶特异性酶解，酶解后离心取上清液。
（2）阴离子交换色谱法提取蛤蟆油中的糖胺聚糖：利用DEAE纤维素树脂的阴离子交换作用，通过不同高盐浓度洗脱液置换出步骤（1）所得蛤蟆油上清液中的糖胺聚糖，洗脱液中的NaCl用透析除去，再冻干浓缩得到蛤蟆油糖胺聚糖OR-GAG。
（3）硫酸软骨素ABC酶酶解蛤蟆油糖胺聚糖获取二糖组分：取步骤(2)中的蛤蟆油糖胺聚糖OR-GAG，加入硫酸软骨素ABC酶，将蛤蟆油糖胺聚糖OR-GAG长链特异性酶解至CS/DS/HA二糖单元。
（4）分子排阻色谱法纯化二糖组分：利用液相分子排阻色谱柱peptide柱分离纯化CS/DS/HA二糖。
（5）LC/MS-ITTOF分析蛤蟆油中CS/DS/HA二糖的结构：酶解得到的CS/DS/HA二糖经AMAC（2-氨基吖啶酮）和NaBH3CN荧光标记后，通过LC/MS-ITTOF分析得到二糖结构及其组分摩尔百分比。
一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法，其特征在于，包括以下具体步骤：
（1）胰蛋白酶酶解蛤蟆油：将蛤蟆油研磨成粉末状并在超纯水中充分浸泡，加入胰蛋白酶酶解蛤蟆油蛋白，酶解结束后离心取上清液。
（2）阴离子交换色谱法提取蛤蟆油中的糖胺聚糖：水洗DEAE柱除去树脂中的乙醇，再加入平衡液使色谱柱达到稳定状态，加入步骤(1)所得上清液，再加入平衡液除去蛋白质杂质，然后用不同高盐浓度洗脱液将蛤蟆油糖胺聚糖(OR-GAG)洗脱出来，收集到的样品进行紫外检测，最后，利用透析除去糖胺聚糖中大量的盐并冻干。
（3）硫酸软骨素ABC酶酶解蛤蟆油糖胺聚糖获取二糖组分：取步骤(2)中的蛤蟆油糖胺聚糖OR-GAG1-100 ng，加入10 μL 30-50 mIU/μL硫酸软骨素ABC，进行酶解，将将蛤蟆油糖胺聚糖OR-GAG长链特异性酶解至CS/DS/HA二糖单元，再用沸水灭活，高速离心取上清液。
（4）分子排阻色谱法纯化二糖组分：取步骤(3)中的上清液，通过分子排阻色谱柱peptide柱对样品进行分离、纯化，从而获得纯净的蛤蟆油CS/DS/HA二糖组分。
（5）LC/MS-ITTOF分析蛤蟆油中CS/DS/HA二糖的结构：用AMAC（2-氨基吖啶酮）和氰基硼氢化钠对蛤蟆油中CS/DS/HA二糖进行荧光标记，标记后的CS/DS/HA二糖用LC/MS-ITTOF分析结构，并根据建立好的标准曲线，得到蛤蟆油中CS/DS/HA的组分含量及摩尔百分比。
上述步骤(1)的具体步骤为：将蛤蟆油在研钵中研磨至粉末状，取100-200 mg粉末状蛤蟆油于20 mL超纯水中常温浸泡24-48 h使其充分溶胀，制成蛤蟆油样品，然后加入100μL 10 μg/uL胰蛋白酶溶液进行酶解，反应条件为36-40℃，24-48 h；酶解结束后，10000rpm离心10-20 min取上清液。
上述步骤(2)的具体步骤为先取5-20 mL水洗过的DEAE树脂装柱，再用十至二十倍柱体积0.2 M NaCl，10-50 mM磷酸平衡缓冲液平衡DEAE柱，待柱稳定后上样，并加入平衡缓冲液除去蛋白杂质，随后利用分段洗脱的方法提取蛤蟆油中的糖胺聚糖，分段洗脱的高盐浓度洗脱液分别为0.5 M NaCl，10-50 mM磷酸缓冲液；1.0 M NaCl，10-50 mM磷酸缓冲液；1.5M NaCl，10-50 mM磷酸缓冲液；2.0 M NaCl，10-50 mM磷酸缓冲液；以3 mL/管收集各馏分，并进行紫外检测，波长为212 nm；DEAE收集到的馏分用1000-5000 Da的透析膜透析除去洗脱液中的NaCl，每两小时换一次去离子水，透析2-4天，最后，冻干2-4天除去水分，得到纯净的蛤蟆油糖胺聚糖(OR-GAG)。
上述步骤(3)的具体步骤为：取步骤（2）获得的蛤蟆油糖胺聚糖OR-GAG1-100 ng，加入10 μL 30-50 mIU/μL硫酸软骨素ABC进行酶解，将蛤蟆油糖胺聚糖OR-GAG酶解至CS/DS/HA二糖单元，酶解条件为36-40℃，酶解24-48 h，，酶解结束后，沸水灭活，10000 r离心10-20min取上清液。
上述步骤(4)的具体步骤为：取步骤3中的上清液，以≤200 μL的上样体积过分子排阻色谱柱peptide柱；CS/DS/HA二糖经peptide柱分离纯化，流动相为0.1-0.3 M NH4HCO3，流速0.4 mL/min，CS/DS/HA二糖在35-44 min出峰并进行收集。流动相中的NH4HCO3通过55-65℃挥发除去。
上述步骤(5)的具体步骤为：用5-10 μL AMAC（2-氨基吖啶酮）荧光标记纯化后的蛤蟆油CS/DS/HA二糖，以上样量<20 μL过反相色谱柱ODS柱，流动相A相为20-80 mM 醋酸铵，pH=5-7，流动相B相为100%甲醇，流速为0.3 mL/ min，CDL为100-200℃，Heat Block为100-200℃，N2流速为1.5 mL/min，检测电压为1.6-1.8 kV。根据建立好的标准曲线，得到二糖组分含量和摩尔百分比。
本发明的优点在于：本发明以蛤蟆油为原料，通过胰蛋白酶酶解蛤蟆油蛋白，阴离子交换色谱法将聚阴离子长链GAG分离出来，再加入硫酸软骨素ABC酶特异性酶解CS/DS/HA长链至二糖单元，用分子排阻色谱法将得到的CS/DS/HA二糖单元进行纯化与分离，最后，CS/DS/HA二糖单元用荧光物质AMAC（2-氨基吖啶酮）进行标记，利用LC/MS-ITTOF对CS/DS/HA二糖单元进行结构分析，得到CS/DS/HA二糖的组分含量和摩尔百分比。本发明的方法为研究蛤蟆油的药用价值提供了化学成分基础，有助于开发蛤蟆油新的保健和药用功能。
附图说明：
图1 DEAE柱分段洗脱所得组分的紫外检测图：C1所对应的的洗脱液浓度为0.5MNaCl，C2所对应的的洗脱液浓度为1.0M NaCl，C3所对应的的洗脱液浓度为1.5M NaCl，C4所对应的的洗脱液浓度为2.0M NaCl。
图2 Peptide柱-HPLC法分离与纯化CS/DS/HA二糖组分图。其中的短横线代表二糖组分的收集时间范围。
图3 CS/DS标准二糖结构。
图4 HA标准二糖结构。
图5 ODS-MS法建立CS/DS/HA二糖标准曲线。
图6蛤蟆油中CS/DS/HA二糖的EIC图：A为8个标准二糖各40 ng的EIC图，B为C1中所含二糖的EIC图，C为C2中所含二糖的EIC图，D为C3中所含二糖的EIC图，E为C4中所含二糖的EIC图。1为2S4S6S，2为2S4S，3为2S6S，4为4S6S，5为2S，6为4S，7为6S，8为0S。
具体实施方式
为了使本发明所述的内容更加便于理解，下面结合具体实施方式对本发明所述的技术方案做进一步的说明，但是本发明不仅限于此。
实施例1
一种从蛤蟆油中提取硫酸软骨素/硫酸皮肤素/透明质酸的方法：
1.胰蛋白酶(Tripsin)酶解蛤蟆油
取0.1 g粉末状蛤蟆油于20 mL超纯水中常温浸泡24 h，充分溶胀后，加入100 μL10 μg/uL胰蛋白酶溶液（胰蛋白酶缓冲为0.2 M NaCl,10 mM NaH2PO4/Na2HPO4，pH=7.5）反应条件为37℃，48 h。酶解结束后，10000 rpm高速离心10 min取上清液。
阴离子交换色谱法提取蛤蟆油中糖胺聚糖
取10 mL水洗过的DEAE树脂装柱，再用10倍柱体积的平衡液将柱子平衡至稳定状态。取步骤1中的上清液上样，用平衡液除去蛋白质等电荷密度低的杂质，再用不同高盐浓度洗脱液提取糖胺聚糖，最后将收集到的样品进行紫外检测，波长为212 nm。紫外检测图见图1。
DEAE提取糖胺聚糖的具体参数如下：
DEAE柱柱体积：10 mL
上样量: 10 mL
收集：3 mL/管
平衡液：0.2 M NaCl,10 mM NaH2PO4/Na2HPO4(pH=7.5)
洗脱液：0.5 M NaCl,10 mM NaH2PO4/Na2HPO4(pH=7.5)，收集样品C1；
1.0 M NaCl,10 mM NaH2PO4/Na2HPO4(pH=7.5)，收集样品C2；
1.5 M NaCl,10 mM NaH2PO4/Na2HPO4(pH=7.5)，收集样品C3；
2.0 M NaCl,10 mM NaH2PO4/Na2HPO4(pH=7.5)，收集样品C4。
柱温：室温
检测器：UV检测器
检测波长：212 nm
3.分子排阻色谱法CS/DS/HA二糖的制备与分离
将C1、C2、C3以及C4四种样品先用3000 Da的透析膜透析三天，将样品中NaCl完全除去，再利用冻干与浓缩除去水分。分别取浓缩干的C1、C2、C3以及C450 ng、25 ng、3 ng、1.5ng分别用硫酸软骨素酶特异性酶解，酶的体积均为10 μL，酶的浓度均为30 mIU/μL，酶缓冲液液为0.1 M NaAc，0.1 mM Ca(Ac)2，pH=7.0（醋酸调pH），反应条件为37℃，24 h。待反应结束后，用100℃沸水灭活并于常温下静置一段时间后，10000 rpm 离心10 min取上清液，上清液以≤200 μL过分子排阻色谱柱peptide柱分离得到CS/DS/HA二糖。
Peptide-HPLC方法具体参数如下：
色谱柱：Superdex™ Peptide 10/300 GL(10 × 300–310 mm)
流速：0.4 mL/min
上样量：200 μL
流动相：0.2 M NH4HCO3
柱温：室温
检测器：UV检测器
检测波长：232 nm
收集时间：35-44 min；
通过上述方法分离CS/DS/HA二糖，得到的液相图见图2。
实施例 2
1.ODS-MS法建立CS/DS/HA二糖标准曲线
取浓度为1 μg/10 μl的8种CS/DS标准二糖各1.3 μg混合均匀并浓缩至无水状态，加入5 μL 0.1 M 2-氨基吖啶酮AMAC （AMAC溶于DMSO:CH3COOH=17:3的溶液中）常温避光涡旋20 min，再加入5 μL 1.0 M NaBH3CN（NaBH3CN溶于超纯水中）水浴45℃反应4 h。反应结束后，用50% DMSO将溶液稀释为80ng/10μL、60ng/10μL、40ng/10μL、20ng/10μL和5ng/10μL，分别进行质谱分析。CS/DS结构见图3，其m/z值见表一，CS/DS标准曲线图见图4。
取10 μg HA二糖浓缩干，加入5 μL 0.1 M AMAC进行荧光标记，反应过程中需用锡箔纸包裹并涡旋20 min，再加入5 μL 1.0 M NaBH3CN发生还原胺化反应，反应条件为45℃，4 h。反应结束后，用50% DMSO将溶液稀释为90ng/10μL、70ng/10μL、50ng/10μL、30ng/10μL和10ng/10μL，进行质谱检测。以二糖质量为横坐标，以峰面积/100000为纵坐标，得到HA二糖的标准曲线为y=2.1052x - 6.1058，R² = 0.9946。HA结构见图4，其m/z值见表一。
ODS-MS方法具体参数如下：
色谱柱：ODS-2 HYPERSIL(4.6×250 mm，5 μm)
流速: 0.3 mL/min
上样量：10 μl
流动相: A相为40 mM 醋酸铵，pH=5.6；B相为100%甲醇
柱温：45℃
CDL和Heat Block温度：150℃
Nebulizing Gas：1.5 mL/min
检测电压：1.8 kV
线性梯度：
0~5 min(15%B)，5~40 min(15%B~60%B)，40~45 min(60%B~100%B)；
通过上述方法得到的标准曲线见图5。
表一 CS/DS/HA标准二糖m/z值
2.LC/MS-ITTOF分析蛤蟆油CS/DS/HA二糖结构
将液相纯化收集的C1、C2、C3和C4组分，55℃挥发NH4HCO3，浓缩干后进行荧光标记，首先加入5 μL 0.1 M AMAC常温避光涡旋20 min，其中，AMAC溶于DMSO:CH3COOH=17:3的溶液中。然后，加入5 μL 1.0 M NaBH3CN（溶于超纯水）水浴45℃，反应4h。最后用50%DMSO稀释后进行质谱分析。得到的质谱图见图6。
根据步骤1所建立的标准曲线，以峰面积/100000为纵坐标，代入曲线得到蛤蟆油中硫酸软骨素、硫酸皮肤素、透明质酸二糖的质量。蛤蟆油C1、C2、C3和C4组分中CS/DS二糖摩尔百分比见表二，蛤蟆油CS/DS/HA总含量见表三。
表二 蛤蟆油中CS/DS/HA二糖组分摩尔百分比
表三 蛤蟆油中CS/DS/HA二糖组分总含量
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
Claims (1)
1.一种提取并检测蛤蟆油中硫酸软骨素/硫酸皮肤素/透明质酸的方法，其特征在于，包括以下步骤：
(1)胰蛋白酶酶解蛤蟆油：将蛤蟆油研磨成粉末状并在超纯水中充分浸泡，加入胰蛋白酶酶解蛤蟆油蛋白，酶解结束后离心取上清液；
(2)阴离子交换色谱法提取蛤蟆油中的糖胺聚糖：水洗DEAE柱除去树脂中的乙醇，再加入平衡液使色谱柱达到稳定状态，加入步骤(1)所得上清液，再加入平衡液除去蛋白质杂质，然后用不同高盐浓度洗脱液将蛤蟆油糖胺聚糖OR-GAG洗脱出来，收集到的样品进行紫外检测，最后利用透析除去糖胺聚糖中大量的盐并冻干；
(3)硫酸软骨素ABC酶酶解蛤蟆油糖胺聚糖获取二糖组分：取步骤(2)中的蛤蟆油糖胺聚糖OR-GAG，加入硫酸软骨素ABC酶，将蛤蟆油糖胺聚糖OR-GAG长链特异性酶解至CS/DS/HA二糖单元，再用沸水灭活，高速离心取上清液；
(4)分子排阻色谱法纯化二糖组分：取步骤(3)中的上清液，通过分子排阻色谱柱peptide柱对样品进行分离、纯化，从而获得纯净的蛤蟆油CS/DS/HA二糖组分；
(5)LC/MS-ITTOF分析蛤蟆油中CS/DS/HA二糖的结构：用2-氨基吖啶酮和氰基硼氢化钠对蛤蟆油中CS/DS/HA二糖进行荧光标记，标记后的CS/DS/HA二糖用LC/MS-ITTOF分析结构，并根据建立好的标准曲线，得到蛤蟆油中CS/DS/HA的组分含量及摩尔百分比；
步骤(1)的具体步骤为：将蛤蟆油在研钵中研磨至粉末状，取100-200mg粉末状蛤蟆油于20mL超纯水中常温浸泡24-48h使其充分溶胀，制成蛤蟆油样品，然后加入100μL 10μg/uL胰蛋白酶溶液进行酶解，反应条件为36-40℃，24-48h；酶解结束后，10000rpm离心10-20min取上清液；
步骤(2)的具体步骤为：先取5-20mL水洗过的DEAE树脂装柱，再用十至二十倍柱体积0.2M NaCl，10-50mM磷酸平衡缓冲液平衡DEAE柱，待柱稳定后上样，并加入所述的平衡缓冲液除去蛋白杂质，随后利用分段洗脱的方法提取蛤蟆油中的糖胺聚糖，分段洗脱的高盐浓度洗脱液分别为0.5M NaCl，10-50mM磷酸缓冲液；1.0M NaCl，10-50mM磷酸缓冲液；1.5MNaCl，10-50mM磷酸缓冲液；2.0M NaCl，10-50mM磷酸缓冲液；以3mL/管收集各馏分，并进行紫外检测，波长为212nm；DEAE收集到的馏分用1000-5000Da的透析膜透析除去洗脱液中的NaCl，每两小时换一次去离子水，透析2-4天，最后，冻干2-4天除去水分，得到纯净的蛤蟆油糖胺聚糖OR-GAG；
步骤(3)的具体步骤为：取步骤(2)中的蛤蟆油糖胺聚糖OR-GAG 1-100ng，加入10μL30-50mIU/μL硫酸软骨素ABC酶进行酶解，将将蛤蟆油糖胺聚糖OR-GAG长链特异性酶酶解至CS/DS/HA二糖单元，酶解温度36-40℃，酶解时间24-48h，酶解结束后，沸水灭活，10000rpm离心10-20min取上清液；
步骤(4)的具体步骤为：取步骤(3)中的上清液，以≤200μL的上样体积过分子排阻色谱柱peptide柱；CS/DS/HA二糖经peptide柱分离纯化，流动相为0.1-0.3M NH4HCO3，流速0.4mL/min，CS/DS/HA二糖在35-44min出峰并进行收集；流动相中的NH4HCO3通过55-65℃挥发除去。
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