CN114634552B - 一种抗肥胖十三肽及其应用 - Google Patents
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Abstract
一种抗肥胖十三肽及其应用，所述肽的序列为SEQ ID NO.1。本申请的肽同时具有显著的胰脂肪酶和胆固醇酯酶抑制功能，且具有安全无毒和良好的人体肠道吸收特性等优点，可应用于生物医药以及功能性食品等领域，具有良好的发展潜力。
Description
技术领域
本申请属于蛋白领域，具体地，本申请提供了一种抗肥胖十三肽及其应用和筛选方法。
背景技术
肥胖是一个全球性的公共卫生问题，其患病率在过去50年中不断增加，因此肥胖问题迫切需要得到解决。目前，临床也在使用一些治疗药物来缓解肥胖。奥利司他是一种有效的胰脂肪酶抑制剂，可通过减少肠道脂肪消化，降低食欲和增加饱腹感来有效减轻体重。然而药物在减肥中可能产生副作用，如恶心、腹泻、便秘、呕吐、消化不良、腹痛。目前生物活性肽因其体积小、体内利用率高、不会产生明显副作用，而被视为常规药物的更好替代品。流行病学研究表明食用豆类与降低超重或肥胖的风险有关。中国是世界上红小豆种植面积最大和产量最高的国家。我们先前的研究表明红小豆热处理蛋白水解物可以缓解高脂喂养小鼠的肥胖，这表明红小豆热处理蛋白水解物可能是体外筛选抗肥胖肽的良好来源。
人体中膳食脂质的消化吸收需要胰脂肪酶和胆固醇酯酶这两种关键酶的参与。因此，抑制这两种关键酶的活性便能减缓脂质的消化吸收，以达到抗肥胖的目的。因此，本发明提供一种来源于红小豆热处理蛋白水解物的对胰脂肪酶和胆固醇酯酶有较好抑制活性的十三肽，该多肽具有抗肥胖能力。
发明内容
针对上述问题，一方面，本申请提供了一种抗肥胖十三肽，所述肽的序列为SEQ IDNO.1。
另一方面，本申请提供了组合物，所述组合物包含上述肽以及药学、食品或保健品上可接受的辅料。
另一方面，本申请提供了上述肽或者组合物在制备胰脂肪酶和/或胆固醇酯酶抑制剂中的用途。
另一方面，本申请提供了上述肽或者组合物在制备治疗肥胖的药物中的应用。
另一方面，本申请提供了上述肽或者组合物在制备适用于肥胖人群的食品或保健品中的应用。
另一方面，本申请提供了筛选上述肽的方法，所述方法包括：
(1)体外模拟消化：使用酶法对红小豆热处理蛋白进行水解以得到蛋白水解物；
(2)降脂肽的筛选：以胰脂肪酶和胆固醇酯酶活性抑制率为评价指标，在红小豆热处理蛋白水解物的不同超滤级分中筛选出具有最佳降脂活性的部分。然后通过质谱测序和虚拟筛选技术找出与胰脂肪酶和胆固醇酯酶对接效果较好的肽段；
(3)抑制效果及机理分析：以胰脂肪酶和胆固醇酯酶活性抑制率为评价指标，对采用Fmoc 固相合成方法制备的抗肥胖肽进行抑制效果评价，并进一步通过分子对接阐明其抑制作用机理。为了解抗肥胖肽更多的特性，最后基于计算机软件评估其毒性、等电点、总平均亲水性和人体肠道吸收性。
进一步地，所述水解使用胃蛋白酶和胰酶。
进一步地，所述超滤级分为＜3kDa、3-10kDa和＞10kDa级分。
有益效果：
本次首次从红小豆中筛选得到能够同时有效抑制胰脂肪酶和胆固醇酯酶活性的功能性多肽FDTGSSFYNKPAG，揭示了该肽与膳食脂质消化酶之间的相互作用，发现了其具有安全无毒和良好的人体肠道吸收特性等优点，可应用于生物医药以及功能性食品等领域，具有良好的发展潜力。
附图说明
图1为不同超滤级分对膳食脂质消化酶活性的抑制结果(A胰脂肪酶抑制率、B胆固醇酯酶抑制率)；
图2为FDTGSSFYNKPAG肽与胰脂肪酶(A)和胆固醇酯酶(B)在活性位点处对接后的最佳位置概述和细节。胰脂肪酶或胆固醇酯酶残基用黄棒模型表示。蓝色、灰色、黄色和绿色虚线分别表示氢键、疏水相互作用、盐桥和π-π堆积
图3为FDTGSSFYNKPAG肽对膳食脂质消化酶活性的抑制结果(A胰脂肪酶抑制率、B胆固醇酯酶抑制率)。
具体实施方式
实施例1红小豆热处理蛋白的制备
红小豆粉使用正己烷进行脱脂，其中固液比为1:3(w/v)，并在过夜静置后弃去正己烷。脱脂红小豆粉和蒸馏水以1:10(w/v)的比例混合，并用1mol/L NaOH将溶液pH值调至8.5，在40℃下连续搅拌1h后，将该溶液在4℃，7000×g下离心30min，收集上清液。用1mol/LHCl将上清液pH值调至4.5后，在室温下静置1h以利于蛋白质沉淀。收集沉淀，用蒸馏水洗涤沉淀三次，并用1mol/L NaOH将蛋白质的pH值调节至7.0。最后，冷冻干燥并保存在 -20℃。对红小豆蛋白进行热处理。简而言之，将蛋白质在沸水中加热10min。
实施例2红小豆热处理蛋白的酶解
将红小豆热处理蛋白按照5％(w/v)的比例在蒸馏水中混合均匀。用1mol/L HCl调节溶液pH值到2.0后添加4％胃蛋白酶(w/w)，并将溶液在37℃下孵育2h。孵育后，先使用0.9mol/L NaHCO3将pH值调至5.3，再用1mol/L NaOH将pH值维持在7.5，然后加入4％胰酶(w/w)。溶液在37℃下孵育2h后，置于沸水浴中10min，终止反应。室温冷却后，在4℃， 13600×g下离心10min，并收集上清液，冷冻干燥，于-20℃保存。
实施例3抗肥胖肽的筛选
超滤可以从蛋白水解物中富集生物活性肽。通过使用10kDa和3kD超滤装置把红小豆热处理蛋白水解物分离成三个不同的级分(>10kDa、3-10kDa和<3kDa)，测定不同级分在4mg/mL浓度下对胰脂肪酶和胆固醇酯酶活性的影响，并冷冻干燥不同级分。结果表明<3kDa级分对膳食脂质消化酶活性抑制效果最好(见图1)。体外酶活性抑制实验具体过程为：
①胰脂肪酶
在96孔酶标板中，将50μL的样品、40μL的2.5mg/mL胰脂肪酶溶液和50μL的 10mM对硝基苯丁酸酯作为底物，在pH 7.3的磷酸缓冲液中37℃孵育30min。酶标仪在405 nm处记录吸光度。使用奥利司他作为阳性对照，并根据公式(1)进行计算。
式(1)中：A：对照的吸光度；B：对照空白的吸光度；C：样品的吸光度；D：样品空白的吸光度。
②胆固醇酯酶
在96孔酶标板中，将50μL的样品、50μL的25μg/mL胆固醇酯酶溶液和50μL 的10mM对硝基苯丁酸酯作为底物，在pH 7.0的磷酸缓冲液(含100mM NaCl，5.16mM牛磺胆酸钠)中25℃孵育5min。酶标仪在405nm处记录吸光度。使用辛伐他汀作为阳性对照，并根据公式(2)进行计算。
式(2)中：A：对照的吸光度；B：对照空白的吸光度；C：样品的吸光度；D：样品空白的吸光度。
随后采用C18除盐柱对<3kDa级分进行除盐，并进一步经由配备在线纳喷离子源的LC-MS/MS完成肽序列的鉴定。采用Dock 6.9对得到的肽序列进行虚拟筛选，根据肽段的对接打分(<-120kcal/mol)筛选得到与胰脂肪酶和胆固醇酯酶对接效果较好的肽段FDTGSSFYNKPAG(见表1)。
表1. FDTGSSFYNKPAG肽与胰脂肪酶和胆固醇酯酶的对接打分
实施例4分子对接
从RCSB Protein Data Bank数据库(http://www.rcsb.org/)获取胰脂肪酶(PDB编号： 1ETH)和胆固醇酯酶(PDB编号：1F6W)的晶体结构，采用Dock 6.9将FDTGSSFYNKPAG与胰脂肪酶和胆固醇酯酶进行半柔性对接，确定其与膳食脂质消化酶作用的关键氨基酸残基及相互作用力(见图2)。与胰脂肪酶对接结果表明，FDTGSSFYNKPAG肽可与9个氨基酸残基相互作用，并且相互作用包括氢键、疏水相互作用、盐桥和π-π堆积。FDTGSSFYNKPAG肽不仅能与催化残基(Ser153)形成氢键，还都能与底物结合残基(Phe78、His152、Phe216、Trp253 和Arg257)产生疏水相互作用和盐桥(见图2A)。与胆固醇酯酶对接结果表明，FDTGSSFYNKPAG肽可与16个氨基酸残基相互作用，并且相互作用包括氢键、疏水相互作用和盐桥。FNTGSSFYNPKAG不仅能与催化残基(Ser194和His435)形成氢键，还能与底物结合残基Ala108 形成疏水相互作用(见图2B)。总之，FDTGSSFYNKPAG通过占据催化位点和底物结合位点来抑制胰脂肪酶和胆固醇酯酶的活性。
实施例5功能评价
FDTGSSFYNKPAG肽由固相合成法制备，并通过高效液相色谱和质谱分析，确定肽纯度大于95％。经体外酶活性抑制实验发现，在4mg/mL的浓度下，FDTGSSFYNKPAG肽的胰脂肪酶和胆固醇酯酶抑制率分别是36.28％和33.75％(见图3)。为了解FDTGSSFYNKPAG肽更多的信息，我们运用计算机软件对其进行了功能预测。FDTGSSFYNKPAG的毒性通过ToxinPred(https://webs.iiitd.edu.in/raghava/toxinpred/index.html)，等电点通过Pepdraw(http://www.tulane.edu/～biochem/WW/PepDraw/)，总平均亲水性通过ExPasy(https://web.expasy.org/protparam/)，人体肠道吸收通过admetSAR (http://lmmd.ecust.edu.cn/admetsar1/home/)评估。如表2所示，FDTGSSFYNKPAG肽是无毒的，具有良好的肠道吸收性。FDTGSSFYNKPAG肽的等电点小于7，表明它呈酸性。总平均亲水性可以用来表征蛋白质的亲疏水性，其中正值越大表明疏水性越强，负值越大表明亲水性越强，故FDTGSSFYNKPAG肽具有更好的亲水性。
表2. FDTGSSFYNKPAG肽的功能预测
SEQUENCE LISTING
<110> 中国农业大学
<120> 一种抗肥胖十三肽及其应用
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 13
<212> PRT
<213> artificail
<400> 1
Phe Asp Thr Gly Ser Ser Phe Tyr Asn Lys Pro Ala Gly
1 5 10
Claims (4)
1.一种抗肥胖十三肽，其特征在于，所述抗肥胖十三肽的序列为SEQ ID NO.1。
2.组合物，其特征在于，所述组合物包含根据权利要求1所述的抗肥胖十三肽以及药学上可接受的辅料。
3.根据权利要求1所述的抗肥胖十三肽或根据权利要求2所述的组合物在制备胰脂肪酶和/或胆固醇酯酶抑制剂中的用途。
4.根据权利要求1所述的抗肥胖十三肽或根据权利要求2所述的组合物在制备治疗肥胖的药物中的应用。
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