CN113429458B - 一种抑制二肽基肽酶iv功能的南极磷虾降血糖寡肽及用途 - Google Patents
一种抑制二肽基肽酶iv功能的南极磷虾降血糖寡肽及用途 Download PDFInfo
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- CN113429458B CN113429458B CN202110885756.9A CN202110885756A CN113429458B CN 113429458 B CN113429458 B CN 113429458B CN 202110885756 A CN202110885756 A CN 202110885756A CN 113429458 B CN113429458 B CN 113429458B
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- dipeptidyl peptidase
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Abstract
本发明公开了一种具有抑制二肽基肽酶IV功能的南极磷虾降血糖寡肽及用途，具体是以南极磷虾为原料，通过超声脱脂、酶解，膜超滤分级和色谱制备得到二肽基肽酶Ⅳ抑制寡肽Leu‑Pro‑Gly‑Thr‑Arg‑Ile‑Pro‑Ala(LPGTRIPA)，ESI‑MS测定分子量为824.0Da。本发明制备的南极磷虾的二肽基肽酶Ⅳ抑制寡肽LPGTRIPA可显著抑制DPP‑Ⅳ活力，显著降低模型小鼠餐后血糖水平，改善小鼠口服葡萄糖糖耐量和麦芽糖糖耐量，降低模型小鼠的甘油三酯(TG)和胆固醇(TC)的含量，且安全无毒副作用，可应用于治疗或者辅助治疗II型糖尿病的特医食品、保健品和药物。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种源于南极磷虾的二肽基肽酶Ⅳ抑制寡肽及其治疗糖尿病的用途。
背景技术
糖尿病是一种以高血糖为特征的代谢性疾病。近年来，我国糖尿病发病率从1980年的0.6％增长到目前的11.6％。国家卫计委最新公布数据表明，我国糖尿病人群已达1.14亿人，其中II型糖尿病接近占糖尿病人群的90％。此外，糖尿病已成为中国慢性肾病(CKD)的首要因素，约20％-40％的糖尿病患者合并慢性肾病，合并症患者的心肾事件风险显著增加，预期寿命显著降低；除此以外，糖尿病并发症还涉及血管、眼、足等多个器官，致残、致死率高，治疗周期长、开销大，给国家、患者及家庭带来沉重的经济负担，成为严重影响我国国民健康的公共问题之一。
胰高血糖素样肽-1(GLP-1)是回肠内分泌细胞分泌的一种脑肠肽，可以通过可以刺激胰岛素、抑制升糖素、抑制胃排空和让胰岛细胞重生的方式来降低血糖。但人体自身产生的GLP-1极易被体内的二肽基肽酶Ⅳ(DPP-Ⅳ)降解，其血浆半衰期不足2分钟。因此，寻找DPP-Ⅳ抑制剂，降低机体分泌的GLP-1的降低速率，维持体内GLP-1的水平，就可以抑制胰高血糖素分泌，从而降低血糖。该方法成为开发治疗糖尿病药物的主攻方向之一。
南极磷虾资源被喻为人类未来的海洋蛋白资源仓库，具有极为重要的开发价值，研究人员利用多种技术指备了系列功能肽，如抗氧化肽、高F值寡肽、降压肽等。例如，吉薇等以南极磷虾为原料制备二肽基肽酶Ⅳ(DPP-Ⅳ)抑制肽AP、IPA和IPAVF。
发明内容
本发明的目的在于提供一种抑制二肽基肽酶IV功能的南极磷虾降血糖寡肽及用途，其可应用于治疗或者辅助治疗II型糖尿病的特医食品、保健品和药物。为达到上述发明目的，采用如下技术方案：
一种南极磷虾寡肽，其特征在于，所述寡肽的氨基酸序列为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)。
优选地，该寡肽为八肽化合物，分子量为824.0Da。
本发明还公开了一种制备上述寡肽的方法，包括如下步骤：
1)南极磷虾预处理：将南极磷虾捣碎后加入乙酸乙酯溶液，脱脂后离心，收集固体沉淀干燥，即得脱脂虾粉；
2)南极磷虾虾粉的酶解：将脱脂虾粉加入到缓冲液中搅匀，溶液调温至45-55℃，加入蛋白酶A，酶解后调溶液pH加入蛋白酶B，酶解后，溶液冷却至室温，离心收集上清液，即得南极磷虾蛋白酶解液；
3)南极磷虾寡肽寡肽的制备：将南极磷虾蛋白酶解液经超滤膜进行分级，收集分级组分，测定各组分对二肽基肽酶Ⅳ的抑制作用，选择活性最好的组分依次经凝胶柱层析和反相高效液相色谱纯化，即得二肽基肽酶Ⅳ抑制寡肽。
优选地，步骤1)包括：
南极磷虾解冻，去头和皮，虾肉组织捣碎机捣碎，按料液比1g:5-20mL加入乙酸乙酯溶液，110-130W超声20-40min脱脂，重复2-5次，于4℃、6000g离心5-20min，固体沉淀干燥，得脱脂虾粉。
更优选地，步骤1)包括：
南极磷虾解冻，去头和皮，虾肉组织捣碎机捣碎，按料液比1g:10-15mL加入乙酸乙酯溶液，120W超声25-30min脱脂，重复3次，于4℃、6000g离心10-15min，固体沉淀干燥，得脱脂虾粉。
优选地，步骤2)包括：
将上述脱脂虾粉按照料液比1g:10-50mL加入到pH 8-9缓冲液中，搅匀，溶液调温至40-60℃，加入脱脂虾粉重量1.0-2.0％蛋白酶A，酶解2-5h；然后调溶液pH至5-6，加入脱脂虾粉重量1.0-2.0％蛋白酶B，酶解2-5h；酶解结束后，溶液冷却至室温，于12000rmp离心15-30min，收集上清液，即南极磷虾蛋白酶解液。
更优选地，步骤2)包括：
将上述脱脂虾粉按照料液比1g:12-15mL加入到pH 8.5缓冲液中，搅匀，溶液调温至45-55℃，加入脱脂虾粉重量1.0-1.5％蛋白酶A，酶解2-3h；然后调溶液pH至5.5-6.5，加入脱脂虾粉重量1.0-1.5％蛋白酶B，酶解2-3h；酶解结束后，溶液冷却至室温，于12000rmp离心20min，收集上清液，即南极磷虾蛋白酶解液。
优选地，蛋白酶A为碱性蛋白酶，酶活力≥1.0×105U/g；蛋白酶B为木瓜蛋白酶，酶活力≥5.0×105U/g。
优选地，步骤3)包括：
将上述南极磷虾蛋白酶解液经截留分子量为5.0和1.0kDa的超滤膜进行分级，收集分级组分，测定各组分对二肽基肽酶Ⅳ(DPP-Ⅳ)的抑制作用(用半数抑制浓度IC50表示)，选择活性最好的组分依次经凝胶柱层析和反相高效液相色谱(RP-HPLC)纯化，得到二肽基肽酶Ⅳ(DPP-IV)抑制寡肽。
本发明还公开了上述寡肽在在制备用于抑制DPP-Ⅳ活力和降低血糖的食品、保健品和药物中的用途。
优选地，南极磷虾寡肽对二肽基肽酶Ⅳ的半抑制浓度IC50为0.029mg/mL。
本发明还公开了上述寡肽在制备治疗和/或辅助治疗和/或预防II型糖尿病和/或减轻II型糖尿病并发症的食品中的用途。
本发明还公开了上述寡肽在制备治疗和/或辅助治疗和/或预防II型糖尿病和/或减轻II型糖尿病并发症的保健品中的用途。
本发明还公开了上述寡肽在制备治疗和/或辅助治疗和/或预防II型糖尿病和/或减轻II型糖尿病并发症的药品中的用途。
与现有技术相比，本发明所提供的南极磷虾寡肽Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)可显著抑制DPP-Ⅳ活力，显著降低糖尿病小鼠餐后血糖水平，改善小鼠口服葡萄糖糖耐量和麦芽糖糖耐量，降低甘油和胆固醇的含量。LPGTRIPA具有安全无毒副作用、降糖活性显著等优点，可应用于治疗或者辅助治疗II型糖尿病的特医食品、保健品和药物。
附图说明
图1为本发明的超滤组分NJH-1的Sephadex LH-20柱层析色谱图；
图2为本发明的Sephadex LH-20柱层析组分NJH-1C的
Peptide 10/300GL分离色谱图；
图3为本发明的
Peptide 10/300GL制备组分NJHS-3的RP-HPLC色谱图；
图4为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)的结构；
图5为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)的质谱图；
图6为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)对糖尿病小鼠餐后血糖曲线下面积的影响；
图7为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)对糖尿病小鼠口服葡萄糖耐量曲线下面积的影响；
图8为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)对糖尿病小鼠口服麦芽糖耐量曲线下面积的影响。
具体实施方式
这里将详细地对示例性实施例进行说明，以下示例性实施例中所描述的实施方式并不代表与本公开相一致的所有实施方式。相反，它们仅是与如所附权利要求书中所详述的、本公开的一些方面相一致的方法的例子。
下述实施例中的实验方法，如无特殊说明，均为常规方法，或按照制造厂商所建议的条件。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
南极磷虾的二肽基肽酶Ⅳ(DPP-Ⅳ)抑制寡肽的制备
制备工艺流程如下：南极磷虾→超声脱脂→酶解→超滤→色谱制备→DPP-Ⅳ抑制寡肽→降糖功能评价。
具体步骤为：
1)南极磷虾预处理：南极磷虾解冻，去头和皮，虾肉组织捣碎机捣碎，按料液比1g:15mL加入乙酸乙酯溶液，120W超声30min脱脂，重复三次，于4℃、6000g离心15min，固体沉淀干燥，得脱脂虾粉；
2)南极磷虾虾粉的酶解：将上述脱脂虾粉按照料液比1g:15mL加入到pH 8.5缓冲液中，搅匀，溶液调温至50℃，加入脱脂虾粉重量1.2％的碱性蛋白酶，酶解2.5h；然后调溶液pH至6.0，加入脱脂虾粉重量1.5％的木瓜蛋白酶，酶解3h，溶液冷却至室温，于12000rmp离心20min，收集上清液，即南极磷虾蛋白酶解液(NJH)。
3)南极磷虾寡肽的制备：将上述酶解液(NJH)经截留分子量5.0和1.0kDa的超滤膜进行分级，收集分级组分NJH-1(MW≤1.0kDa)、NJH-2(1.0≤MW≤5.0kDa)和NJH-3(5.0≤MW)，测定其对DPP-Ⅳ的抑制作用(见表1)。
表1南极磷虾蛋白酶解物和分离各组分的二肽基肽酶Ⅳ(DPP-Ⅳ)抑制活性
| IC<sub>50</sub>(mg/mL) | IC<sub>50</sub>(mg/mL) | ||
| NJH | 1.552 | NJH-1C | 0.394 |
| NJH-1 | 0.536 | NJH-1D | 0.994 |
| NJH-2 | 1.723 | NJHS-1 | 1.046 |
| NJH-3 | ＞5.0 | NJHS-2 | 0.465 |
| NJH-1A | 1.681 | NJHS-3 | 0.157 |
| NJH-1B | 0.82 | NJHS-4 | 0.482 |
由表1可知，活性最好的组分为NJH-1，因此选择NJH-1依次经凝胶色谱和反相高效液相色谱(RP-HPLC)纯化，得到高活性DPP-Ⅳ抑制寡肽，利用氨基酸序列分析仪和质谱测定其结构，具体过程为：
①凝胶色谱层析：将上述NJH-1溶于双蒸水配成浓度为50mg/mL的溶液，经过葡聚糖凝胶Sephadex LH-20柱(2.6×80cm)层析分离，用双蒸水进行洗脱，流速0.5mL/min，根据220nm下的吸光度制作凝胶层析色谱图收集各色谱峰NJH-1A-D(见图1)，测定各色谱峰对DPP-Ⅳ的抑制作用(见表1)，由表可知活性最好的组分是NJH-1；将NJH-1C溶于双蒸水，配成浓度为25mg/mL的溶液，经过
Peptide 10/300GL分离，用双蒸水进行洗脱，流速0.8mL/min，根据220nm下色谱图收集各色谱峰NJHS-1-4(见图2)，测定各色谱峰的DPP-Ⅳ抑制作用(IC50)，活性最好的为NJHS-3，因此取凝胶层析酶解物NJHS-3进行后续实验。
②RP-HPLC精制：将上述NJHS-3用双蒸水配成浓度为50μg/mL的溶液，用RP-HPLC进行纯化(进样量10μL；色谱柱Kromasil C-18(250mm×4.6mm，5μm)；流动相50％乙腈；紫外检测波长220nm，根据220nm下的吸光度曲线收集寡肽NJP1-6(见图3)，测定寡肽对DPP-Ⅳ抑制作用(见表2)。
表2南极磷虾寡肽对二肽基肽酶Ⅳ(DPP-Ⅳ)抑制活性
| IC<sub>50</sub>(mg/mL) | IC<sub>50</sub>(mg/mL) | ||
| NJP1 | 0.062 | NJP4 | 0.267 |
| NJP2 | 0.137 | NJP5 | 0.029 |
| NJP3 | 0.067 | NJP6 | 0.174 |
由表2可知DPP-Ⅳ抑制寡肽NJP5活性最高，因此取NJP5进行后续试验。
试验例1
DPP-Ⅳ抑制寡肽结构检测
收集活性最高的NJP5，采用Edman降解法，用ABI 494蛋白/多肽测序仪测定氨基酸序列为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala(LPGTRIPA)(见图4)，ESI-MS测定分子量为824.0Da(见图5)。
试验例2
DPP-Ⅳ抑制寡肽功能评价
一、糖尿病小鼠模型的准备
1.取SPF级昆明小鼠适应性饲养一周，随机分成高脂组和空白组；
2.模型组采用高脂饲料喂养，空白组采用普通饲料喂养，两组均自由饮水；
3.高脂饲养4周后禁食6h后测定小鼠体重以及血糖；
4.给高脂组小鼠腹腔注射100mg/kg的链脲佐菌素后继续采用高脂饲料饲喂；
5.一周后再次测定小鼠体重以及血糖，空腹血糖在11.0mM以上为糖尿病小鼠；
6.取造模成功的糖尿病小鼠，随机分为模型组、阿卡波糖组、LPGTRIPA组，分别给予生理盐水、阿卡波糖、LPGTRIPA，空白组给予生理盐水作为对照；给药共持续四周。
二、DPP-Ⅳ抑制寡肽对小鼠餐后血糖水平的影响
使用血糖仪检测小鼠餐后血糖浓度，具体步骤如下：
每次小鼠给药后再投喂饲料，进食结束后2h尾静脉取血测定血糖浓度并统计血糖变化曲线下面积(见图6)。
由图6可知，LPGTRIPA能显著降低餐后血糖水平。
三、DPP-Ⅳ抑制寡肽对小鼠口服葡萄糖糖耐量和麦芽糖糖耐量的影响
在给药的第三周和第四周对小鼠口服葡萄糖糖耐量和麦芽糖糖耐量进行测试，具体步骤如下：
测定小鼠体重和空腹血糖后给药，给药后1h再给予各组小鼠2.5g/kg的葡萄糖和2.0g/kg的麦芽糖并在30、60、90、120min时尾静脉取血测定血糖浓度并统计血糖变化曲线下面积(见图7、8)。
由图7和8可知，LPGTRIPA能明显改善小鼠口服葡萄糖糖耐量和麦芽糖糖耐量。
四、DPP-Ⅳ抑制寡肽对小鼠血清中甘油(TG)和胆固醇(TC)的含量的影响
取给药后的小鼠，禁食过夜后眼球取血并加入肝素钠溶液保存血液，5000rpm离心10min后取上清并使用甘油三酯(TG)试剂盒(上海碧云天生物工程研究所)和胆固醇(TC)试剂盒(上海碧云天生物工程研究所)按说明书操作检测TG和TC含量，检测结果见表3。
表3南极磷虾寡肽LPGTRIPA对糖尿病小鼠甘油三酯(TG)和胆固醇(TC)含量的影响
| 组别 | 剂量(mg/kg) | TG(mmol/mL) | TC(mmol/mL) |
| 空白组 | -- | 1.55 | 5.23 |
| 模型组 | -- | 3.08 | 9.75 |
| 阿卡波糖组 | 100 | 2.96 | 9.31 |
| LPGTRIPA | 100 | 2.77 | 9.03 |
由表3可知，LPGTRIPA可以显著降低甘油三酯和胆固醇的含量
以上所述仅为本发明的优选实施例，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
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<110> 浙江海洋大学
<120> 一种抑制二肽基肽酶 IV 功能的南极磷虾降血糖寡肽及用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Leu Pro Gly Thr Arg Ile Pro Ala
1 5
Claims (8)
1.一种南极磷虾寡肽，其特征在于，所述寡肽的氨基酸序列为Leu-Pro-Gly-Thr-Arg-Ile-Pro-Ala。
2.如权利要求1所述的寡肽，其特征在于，所述寡肽的分子量为824.0 Da。
3.如权利要求1所述的寡肽，其特征在于，所述寡肽具有二肽基肽酶Ⅳ抑制活性。
4.一种制备如权利要求1所述寡肽的方法，包括如下步骤：
1）南极磷虾预处理：将南极磷虾捣碎后加入乙酸乙酯溶液，脱脂后离心，收集固体沉淀干燥，即得脱脂虾粉；
2）南极磷虾虾粉的酶解：将脱脂虾粉加入到缓冲液中搅匀，溶液调温至45-55℃，加入蛋白酶A，酶解后调溶液pH加入蛋白酶B，酶解后，溶液冷却至室温，离心收集上清液，即得南极磷虾蛋白酶解液；其中，所述蛋白酶A为碱性蛋白酶，酶活力≥1.0×105 U/g；蛋白酶B为木瓜蛋白酶，酶活力≥5.0×105 U/g；
3）南极磷虾寡肽的制备：将南极磷虾蛋白酶解液经超滤膜进行分级，收集分级组分，测定各组分对二肽基肽酶Ⅳ的抑制作用，选择活性最好的组分依次经凝胶柱层析和反相高效液相色谱纯化，即得二肽基肽酶Ⅳ抑制寡肽。
5.权利要求1所述寡肽在制备用于抑制DPP-Ⅳ活力和降低血糖的药物中的用途。
6.如权利要求5所述用途，其特征在于，所述南极磷虾寡肽对二肽基肽酶Ⅳ的半抑制浓度IC50为0.029mg/mL。
7.权利要求1所述寡肽在制备辅助降血糖的保健品中的用途。
8.权利要求1所述寡肽在制备治疗和/或辅助治疗和/或预防II型糖尿病和/或减轻II型糖尿病并发症的药品中的用途。
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