CN114106128A - 具有α-淀粉酶抑制活性的小米醇溶蛋白肽 - Google Patents
具有α-淀粉酶抑制活性的小米醇溶蛋白肽 Download PDFInfo
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Abstract
本申请提供了三种具有α‑淀粉酶抑制活性的小米醇溶蛋白肽PFQQCH、TPNFF和FMLPQ，其在制备糖尿病患者适用的食品、保健品、药物中的应用以及相应产品。本申请首次从小米醇溶蛋白中筛选得到了能够有效抑制α‑淀粉酶活性的小肽，并明确了其结构；同时PFQQCH、TPNFF和FMLPQ具有安全无毒副作用和良好的人体肠道吸收特性等优点，因此作为功能成分用于食品、保健品以及降糖药品中，具有良好的潜力和应用前景。
Description
技术领域
本申请属于生物技术领域和糖尿病治疗领域。具体地，本申请提供了三种具有α-淀粉酶抑制活性的小米醇溶蛋白肽，其在制备糖尿病患者适用的食品、保健品、药物中的应用以及相应产品。
背景技术
淀粉是人体饮食中最重要的碳水化合物来源之一，约占人类饮食能量的40-60％。然而，体内淀粉的快速消化会导致餐后血糖快速升高，这与2型糖尿病呈正相关。因此，如何减缓淀粉消化受到了广泛关注。目前有两种方法可以调节淀粉消化。一是改变淀粉的理化性质，如物理改性、化学改性或生物改性，二是降低与淀粉水解相关的消化酶的活性，如α-淀粉酶。通常，淀粉会被α-淀粉酶消化成麦芽糖和糊精，随后进一步转化为葡萄糖，从而增加血糖水平。因此，抑制α-淀粉酶是缓解餐后血糖的有效方法。
目前应用于临床上用来控制糖尿病餐后血糖的α-淀粉酶抑制剂会引起许多不良反应，如腹泻、腹部不适、气胀和肠胃胀气。因此，有必要寻找能够治疗2型糖尿病且无副作用或副作用风险低的新型α-淀粉酶抑制剂。近些年来，食物蛋白质作为功能肽的主要来源被广泛研究。许多食源性肽已被证明具有抗菌特性、降血压作用、降低胆固醇、抗血栓形成和抗氧化活性等。小米中的醇溶蛋白能够发挥较好的降糖功效。目前已从小米蛋白中分离出了抗氧化、抗炎以及脂肪酶抑制肽，尚未有关于小米蛋白降糖肽的相关研究。小米醇溶蛋白在通过灌胃被给予糖尿病小鼠后，经过胃肠道消化被水解为氨基酸和肽，进入血液循环发挥包括降糖等生理作用。因此，小米醇溶蛋白水解得到的小分子肽可以作为潜在的功能性降糖物质。但目前对小米中具有改善血糖作用的机制的活性组分不清。
发明内容
一方面，本申请提供了具有α-淀粉酶抑制活性的小米醇溶蛋白肽，其由小米醇溶蛋白消化而成。
进一步地，所述小米醇溶蛋白肽氨基酸序列为:
PFQQCH，SEQ ID NO.1；
TPNFF，SEQ ID NO.2；
或者FMLPQ，SEQ ID NO.3。
进一步地，所述小米醇溶蛋白肽氨基酸序列为:TPNFF，SEQ ID NO.2。
另一方面，本申请提供了上述小米醇溶蛋白肽作为α-淀粉酶抑制剂的应用。
另一方面，本申请提供了上述小米醇溶蛋白肽在制备治疗糖尿病的药物，或者糖尿病或者适用的食品或保健品中的应用。
另一方面，本申请提供了一种药物组合物，其包含上述小米醇溶蛋白肽。
进一步地，所述药物组合物为口服剂型。
进一步地，所述药物组合物还包含药学上可接受的赋形剂。
另一方面，本申请提供了一种食品或保健品，其包含上述小米醇溶蛋白肽。
本申请中的药物、保健品或食品中除本申请的小米醇溶蛋白肽之外还可以包含药品、食品、保健品中可以接受的各种辅料或者赋形剂，所述辅料或者赋形剂包括但不限于填充剂、矫味剂、pH调节剂、缓冲剂、稳定剂、抗氧化剂等。
本申请的小米醇溶蛋白肽可以与其他本领域已知或研究中的有α-淀粉酶抑制活性或者糖尿病治疗作用/辅助治疗的中西药物或保健品联合使用，这些中西药物或保健品可以与本申请的药物、食品、保健品混合、处于一个包装中或者在独立的包装中。
本申请中的糖尿病包括但不限于1型和2型糖尿病。
有益效果为：
本申请首次从小米醇溶蛋白中筛选得到了能够有效抑制α-淀粉酶活性的三条未经报道的小肽PFQQCH、TPNFF和FMLPQ，并明确了小肽PFQQCH、TPNFF和FMLPQ的结构；同时PFQQCH、TPNFF和FMLPQ具有安全无毒副作用和良好的人体肠道吸收特性等优点，因此作为功能成分用于食品、保健品以及降糖药品中，具有良好的潜力和应用前景。
附图说明
图1为PFQQCH与α-淀粉酶的对接结果的3d图；
图2为TPNFF与α-淀粉酶的对接结果的3d图；
图3为FMLPQ与α-淀粉酶的对接结果的3d图；
图4为三种多肽的α-淀粉酶抑制率柱状图。
具体实施方式
实施例1蛋白的体外模拟消化酶解及虚拟筛选
本发明采用体外模拟消化的方法对小米醇溶蛋白进行酶解，将酶切后产生的小米醇溶蛋白水解物进行超滤处理，得到小于3kDa,大于3kDa且小于10kDa,和大于10kDa的肽组分，通过质谱对小于3kDa的肽组分测序，得到置信度较高的肽段序列。然后采用殷赋云平台(http://cloud.yinfotek.com/)中的Dock 6.9对得到的肽段序列进行虚拟筛选，根据肽段的格点打分(<-70kcal/mol)以及内部排斥能(<20kcal/mol)筛选得到与α-淀粉酶对接效果较好的肽段。具体的：
小米醇溶蛋白的提取：
采用盐析法提取小米醇溶蛋白。取脱脂小米粉，加入70％乙醇(v/v)，料液比为1：7，在50℃条件下水浴振荡2小时，在7000rpm下离心20min并收集上清液，加入0～4℃的冷盐水稀释至原体积的3倍，缓慢加入并搅拌，使得最终溶液中氯化钠浓度为0.3％，在0-4℃条件下静置1d使蛋白沉淀。然后在7000rpm下离心20min并收集沉淀，冷冻干燥后即为小米醇溶蛋白，蛋白纯度为90％
小米醇溶蛋白的体外模拟消化：
将小米蛋白样品与蒸馏水以1：5的比例混合，调节pH至3,加入2000U/mL的胃蛋白酶，反应2h后调节pH到7，再加入胰酶使得消化液中胰酶活力为100U胰蛋白酶/mL消化液，继续反应2h，反应结束后沸水浴灭酶10min以终止反应。最后在7000rpm下离心10min收集沉淀,得到蛋白消化后的水解产物。
小米醇溶蛋白体外模拟消化产物的超滤：
用超纯水预清洗
Ultra-15离心过滤器，洗后甩干。然后选取分离分子量为3kDa的离心过滤器，向过滤器内加入不超过15mL的样本，将盖好盖子的过滤装置放入离心转子中，转速为5000×g，旋转约30min。离心结束后，取下盖子和过滤器，将离心管中的液体收集并冻干，即为小于3kDa的蛋白水解物超滤样本。
小米醇溶蛋白体外模拟消化产物的质谱测序及筛选：
采用电喷雾-组合型离子阱Orbitrap质谱仪对小于3kDa的肽组分进行质谱测序，采用De novo的方法进行多肽序列解析，得到肽序列。然后基于格点打分小于-70kcal/mol和内部排斥能小于20kcal/mol对测序得到的肽段进行筛选，筛选结果如表1所示。
表1虚拟筛选中与α-淀粉酶对接效果较好的肽段
实施例2靶向筛选
利用PeptideRanker在线系统筛选得到的具有较好生物活性的肽段(http://bioware.ucd.ie/～compass/biowareweb/Server_pages/peptideranker.Php)，评分大于0.5则表示可能具有生物活性。然后通过在线工具Expasy和admetSAR对筛选得到的生物活性较好的肽段进行水溶性、不稳定性、等电点、半衰期和ADMET性质的预测，筛选得到可能具有生物活性的肽段。
实施例3活性位点分析和功能预测
从PDB数据库(http：//www.rcsb.org/)中获得人的α-淀粉酶晶体结构(PDB编号：5E0F)，并将其作为蛋白靶标。然后采用殷赋云平台(http://cloud.yinfotek.com/)中的小工具：准备多肽结构来合成筛选得到的具有生物活性的多肽序列的3D结构。使用殷赋云平台中的Dock 6.9软件进行筛选得到的肽序列与α-淀粉酶的对接，确定其与α-淀粉酶作用的关键氨基酸及相互作用力。
继续使用殷赋云平台中的Dock 6.9软件进一步进行精确分子对接分析，找出PFQQCH、TPNFF和FMLPQ与酶作用的活性位点，如图1-3所示。PFQQCH与α-淀粉酶的残基Tyr151和Ile 235形成疏水相互作用，与Lys 200、His 305和Glu 233形成氢键，与Arg 195和His 299形成盐桥。PFQQCH与α-淀粉酶的残基Leu 165、Leu 162、Trp 58和Ile 235形成疏水相互作用，与His 299、Glu 233和Trp 59形成氢键,与Trp 62形成π-阳离子相互作用，与Glu233和Asp 197形成盐桥。FMLPQ与α-淀粉酶的残基Leu 165、Leu 162、Trp 59、Trp 62、Gln63和Asp 300形成疏水相互作用，与Asp 197和Lys 200形成盐桥，与Gly 238形成氢键。PFQQCH、TPNFF和FMLPQ的稳定性通过以下在线工具(http://web.expasy.org/protparam/)评估。利用admetSAR预测PFQQCH、TPNFF和FMLPQ的ADMET(http://lmmd.ecust.edu.cn/admetsar1/predict/)性质，主要包括人体肠道吸收(human intestinal absorption，HIA)和急性口服毒性(Acute Oral Toxicity)，如表2所示。可以看到，PFQQCH、TPNFF和FMLPQ三条肽段均具有较好的生物活性、人体肠道吸收和低毒性。其中TPNFF具有最好的亲水性、体外稳定性、半衰期性能。
表2肽段PFQQCH、TPNFF和FMLPQ的体外功能预测分析
实施例4三种肽的实际活性验证
我们在体外验证了三种肽的实际α-淀粉酶抑制活性，具体方法参考Wongsa等[1]的方法测定。500μL样品(0.5％)和500μL 0.02mol/L磷酸钠缓冲液(pH＝6.9，0.006mol/LNaCl，包括α-淀粉酶溶液(13U/mL))在25℃下孵育10min，预孵育后加入500μL 1％可溶性淀粉溶液(0.02mol/L磷酸钠缓冲液，pH＝6.9，0.006mol/L NaCl)，在25℃下孵育10min，并加入1.0mL DNS试剂，之后将上述溶液在沸水浴中煮沸5min，停止反应并冷却到室温。反应液用10mL蒸馏水稀释并在540nm处读数。对照选用去离子水替换样品。结果如图4所示，显示三种淀粉酶在浓度为2.5mg/ml的条件下均有明显的α-淀粉酶抑制活性，其中TPNFF的抑制活性较高，达到约35％。
[1]Wongsa P,Chaiwarit J,Zamaludien A.In vitro screening of phenoliccompounds,potential inhibition againstα-amylase andα-glucosidase of culinaryherbs in Thailand[J].Food Chemistry,2012,131(3):964-971.
SEQUENCE LISTING
<110> 中国农业大学
<120> 具有α-淀粉酶抑制活性的小米醇溶蛋白肽
<160> 3
<170> PatentIn version 3.5
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Claims (9)
1.具有α-淀粉酶抑制活性的小米醇溶蛋白肽，其特征在于，其由小米醇溶蛋白消化而成。
2.根据权利要求1所述的小米醇溶蛋白肽，所述小米醇溶蛋白肽氨基酸序列为:
PFQQCH，SEQ ID NO.1；
TPNFF，SEQ ID NO.2；
或者FMLPQ，SEQ ID NO.3。
3.根据权利要求2所述的小米醇溶蛋白肽，其中所述小米醇溶蛋白肽氨基酸序列为:TPNFF，SEQ ID NO.2。
4.根据权利要求1-3任一项所述的小米醇溶蛋白肽作为α-淀粉酶抑制剂的应用。
5.根据权利要求1-3任一项所述的小米醇溶蛋白肽在制备治疗糖尿病的药物，或者糖尿病或者适用的食品或保健品中的应用。
6.一种药物组合物，其包含根据权利要求1-3任一项所述的小米醇溶蛋白肽。
7.根据权利要求6所述的药物组合物，其中所述药物组合物为口服剂型。
8.根据权利要求6或7所述的药物组合物，其中所述药物组合物还包含药学上可接受的赋形剂。
9.一种食品或保健品，其包含上述根据权利要求1-3任一项所述的小米醇溶蛋白肽。
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| CN114716523A (zh) * | 2022-04-15 | 2022-07-08 | 中国农业大学 | 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 |
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| 姬中伟: "小米醇溶蛋白肽的制备及其抗氧化与抗炎活性研究", 《《中国博士学位论文全文数据库 工程科技Ⅰ辑》》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114716524A (zh) * | 2022-04-15 | 2022-07-08 | 中国农业大学 | 抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽 |
| CN114716523A (zh) * | 2022-04-15 | 2022-07-08 | 中国农业大学 | 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 |
| CN114716523B (zh) * | 2022-04-15 | 2023-05-23 | 中国农业大学 | 具有α-葡萄糖苷酶抑制活性的小米醇溶蛋白肽 |
| CN114716524B (zh) * | 2022-04-15 | 2023-05-23 | 中国农业大学 | 抑制α-淀粉酶和α-葡萄糖苷酶的熟小米醇溶蛋白肽 |
| US11976098B2 (en) | 2022-04-15 | 2024-05-07 | China Agricultural University | Cooked millet prolamin peptide for inhibiting alpha-amylase and alpha-glucosidase |
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