CN110105431B - 一种芝麻多肽及其提取方法和在制备抗氧化和/或降血压药物中的应用 - Google Patents
一种芝麻多肽及其提取方法和在制备抗氧化和/或降血压药物中的应用 Download PDFInfo
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Abstract
本发明属于农副产品深加工技术领域，具体涉及一种芝麻多肽，该多肽由九个氨基酸组成，其序列为：Ser‑Tyr‑Pro‑Thr‑Glu‑Cys‑Arg‑Met‑Arg。本发明芝麻多肽可以通过化学固相合成方法人工合成，或者也可以经酶解芝麻蛋白后分离纯化获得。本发明通过测定DPPH清除能力、ABTS清除能力及ACE抑制能力发现：该多肽具有强抗氧化与强降血压活性，可以作为功能活性成分用于制备药物、保健品、食品、营养强化剂、动物饲料、化妆品、日化用品等领域，具有广阔的市场前景。
Description
技术领域
本发明属于农副产品深加工技术领域，具体涉及一种来源于芝麻蛋白的活性多肽SYPTECRMR，以及制备方法和其在制备具有抗氧化和/或降血压药物中的应用。
背景技术
研究证实，由自由基引发的氧化性应激会造成失明、关节炎、阿尔兹海默症、心血管疾病等200多种非传染慢性疾病的发生发展，严重危害人体健康。另外，高血压已经成为严重危害我国居民健康的非传染慢性疾病，我国成人高血压患病人数达2.45亿，高血压直接经济负担占我国卫生总费用的6.6%。因此，开发抗氧化与降血压的功能因子具有重要的社会意义和广阔的市场价值。
低值蛋白经过适当酶解可以产生高附加值抗氧化肽与降血压肽。抗氧化肽具有延缓机体衰老的功能活性，不仅能够清除体内的自由基，减轻自由基对机体的损伤，而且能够防止紫外线引起的线粒体的损伤和自由基诱导的脂质过氧化；降血压肽是通过抑制体内血管紧张酶的活性，从而起到抑制血压上升的作用。较人工合成的抗氧化剂与降血压药，抗氧化肽与降压肽具有作用平缓、安全性高等优点，具有极高的开发应用价值。
我国芝麻蛋白资源丰富，据测算，每年能产生大约30万吨芝麻蛋白。由于加工技术落后，芝麻蛋白资源未得到充分利用，造成资源浪费。前人研究发现，芝麻蛋白经过酶解可以产生抗氧化肽、降血压肽。若能制备同时具有抗氧化与降血压活性的芝麻活性肽，则更具有市场竞争力，应用范围更加广泛，可以有效解决芝麻蛋白附加值低的问题，推动芝麻蛋白资源化利用。
发明内容
针对人工合成抗氧化剂与降血压药有毒副作用，且目前尚未报道一种同时具有抗氧化和/或降血压活性的多肽，本发明目的在于提供一种来自芝麻蛋白的具有抗氧化和/或降血压活性的多肽，该芝麻多肽具有活性高、制备工艺成熟，成本可控等优点，具有广阔的市场前景。
本发明还提供了上述芝麻多肽的制备方法，以及其在制备具有抗氧化和/或降血压药物方面的应用。
为实现上述目的，本发明采用以下技术方案：
本发明提供一种具有抗氧化和/或降血压活性功效的芝麻多肽，该多肽由九个氨基酸组成，其序列为：Ser-Tyr-Pro-Thr-Glu-Cys-Arg-Met-Arg（SYPTECRMR）。
上述的芝麻多肽，其可以通过人工化学合成制备，如采用固相合成法按照本领域常规工艺制备合成。
或者，该芝麻多肽来源于芝麻2S储藏前体蛋白1（2SS1_SESIN）。本发明还提供了另外一种从芝麻蛋白中提取上述芝麻多肽的方法，其通过蛋白酶解、分离纯化等步骤制得；具体包括下述步骤：
1）芝麻蛋白提取：
将除杂后的芝麻粉碎、脱脂后，提取蛋白，然后离心、取上清液浓缩，获得蛋白提取液，4-10℃低温备用；
2）蛋白酶解：
将步骤1）所得蛋白提取液的pH值调至8.0-9.0，加入蛋白提取液0.05-1%（w/v）的复合蛋白酶，于30-50℃酶解1-5h，酶解结束后中和酶解液，离心取上清液，4-10℃低温备用；
3）分离纯化：
将步骤2）所得上清液用截留分子量2000-10000 Da的超滤膜进行超滤，透过液采用纳滤进行脱盐，然后采用制备液相色谱分离纯化，收集高活性组分，即收集保留时间在21min-22.5 min的组分，冷冻干燥。将冷冻干燥获得的多肽样品，通过Nano-LC-ESI-MS/MS进行分析鉴定，鉴定具有抗氧化与降血压活性的多肽的氨基酸序列为：Ser-Tyr-Pro-Thr-Glu-Cys-Arg-Met-Arg。
具体的，步骤1）中芝麻脱脂采用溶剂萃取法，采用石油醚、正已烷或乙醚进行脱脂；提取蛋白时采用蒸馏水，料液比（w/v）为1:5-25，室温搅拌30-120 min，pH为6-8。上清液浓缩时可以采用旋转蒸发或膜分离，其中膜分离采用的超滤膜截留分子量在5000-100000Da。
具体的，步骤2）中所述复合蛋白酶由碱性蛋白酶Alcalase、碱性蛋白酶2709和胰蛋白酶中的一种或两种以上组成的混合物。
进一步优选的，步骤3）中制备液相色谱的条件为：流动相为含0.1%三氟乙酸的55%甲醇水溶液，即55%甲醇溶液（含0.1%三氟乙酸）；流速8 mL，检测波长为220nm，色谱柱为AQ-C18（300×20mm i.d., 5 μm）。
本发明还提供了上述芝麻多肽作为主要功能成分在制备抗氧化和/或降血压药物、保健品、食品、营养强化剂、动物饲料、化妆品、日化用品等领域中的应用。
与现有技术相比，本发明具有如下优点：
1）本发明所用的原料为芝麻，来源广泛，且提取蛋白后，不影响其他组分的后续利用开发，因此具有成本可控，原料利用率高，对环境友好的优点；
2）本发明提供的芝麻活性多肽，分子量小，易于人体吸收，且具有抗氧化肽和/或降血压活性功效；
3）本发明提供的芝麻多肽活性显著，不仅可以作为食品添加剂，也可以作为功能性成分应用于药物、保健品、食品、营养强化剂、动物饲料、化妆品及日化用品中。
附图说明
图1为实施例1制备液相分离超滤透过液的色谱图；
图2为实施例1提取所得芝麻多肽样品在Nano-LC-ESI-MS/MS中的二级质谱图；
图3为实施例2中人工合成活性多肽的HPLC图；
图4为实施例2中人工合成活性多肽的质谱图；
表1为实施例1和2制备所得多肽SYPTECRMR的DPPH清除、ABTS清除、ACE抑制的IC50。
具体实施方式
以下结合实施例和附图，对本发明的技术方案作进一步地详细介绍，但本发明的保护范围并不局限于此。基于本发明中的实施例，本领域普通技术人员未做出创造性劳动前提下所获得的其他实施例，都属于本发明的保护范围。
实施例1
一种从芝麻蛋白中提取芝麻多肽的方法，其具体包括如下步骤：
1）芝麻蛋白提取：
取1 kg除去石子、草籽等杂质后的芝麻，粉碎至40目，采用索氏抽提器进行脱脂，脱脂条件为：正已烷为萃取剂，温度为70℃，脱脂6h。将脱脂后的芝麻按料液比（w/v）1:15加入蒸馏水，pH调节为7.5，室温搅拌90 min进行蛋白提取。提取结束后，5000 rpm离心15min，取上清液，采用截留分子量为10000Da的超滤膜进行浓缩，浓缩至上清液原体积的10%终止浓缩，收集浓缩液即为蛋白提取液，4-10℃低温备用。
2）蛋白酶解：
将步骤1）所得蛋白提取液的pH值调节到8.5，加入蛋白提取液1%（w/v）的复合蛋白酶（由碱性蛋白酶Alcalase和胰蛋白酶组成，两者质量比为3:1），设定于45℃酶解3 h，酶解结束后，用5 mol/L HCl中和酶解液至pH为7，8000 rpm离心10 min，收集上清液，4-10℃低温备用。
3）分离纯化
将步骤2）所得上清液，采用截留分子量3000 Da的超滤膜进行超滤，透过液采用纳滤（纳滤膜截留分子量为200 Da）进行脱盐，然后采用制备液相色谱分离纯化，制备液相色谱的条件为：流动相为55%甲醇溶液（含0.1%三氟乙酸），流速8 mL，检测波长为220nm，色谱柱为AQ-C18(300×20mm i.d., 5 μm)，收集保留时间在21min-22.5 min的组分（见图1），-60℃冷冻干燥24 h，获得芝麻多肽样品。
将冷冻干燥获得的芝麻多肽样品，通过Nano-LC-ESI-MS/MS进行分析鉴定（见图2），鉴定该多肽的氨基酸序列为：Ser-Tyr-Pro-Thr-Glu-Cys-Arg-Met-Arg（SYPTECRMR）。
实施例2
一种人工合成芝麻多肽的方法，其采用化学固相合成法人工合成。基本流程如下：首先将一个氨基被Fmoc基团保护的氨基酸连接在不溶性固相载体Wang树脂上，然后脱掉氨基的保护基，第一个氨基酸即连接到固相载体上了；其次将氨基被Fmoc基团保护的第二个氨基酸的羧基用缩合剂活化，活化后的氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键，此时在固相载体上就生成了一个带有保护基的二肽。重复上述的肽键形成反应，使肽链从C端向N端生长，直至达到所需要的肽链长度，最后切割得到目的多肽Ser-Tyr-Pro-Thr-Glu-Cys-Arg-Met-Arg（SYPTECRMR）。合成的芝麻多肽序列的液相质谱分析图见图3和图4，此高纯度合成肽的主要离子峰质荷比为1142.32，符合需要合成序列的分子量，说明固相合成成功。
上述采用固相合成法合成芝麻多肽的具体过程参见：①黄惟德、陈常庆著，多肽合成，科学出版社，1985年。②.N.休厄德、H.D.贾库布克著，刘克良等译，肽：化学与生物学，科学出版社，2005年。），该芝麻多肽也可以委托相应的多肽生物公司进行合成，因其化学合成过程不是本申请的重点所在，故本申请此处不再对化学合成过程进行详细赘述。
抗氧化与降血压活性应用试验
下述应用试验中，DPPH、ABTS清除能力及ACE抑制能力的测定参照如下进行。多肽溶液为将冻干或人工合成的芝麻多肽，加入蒸馏水稀释，配成一定浓度的水溶液。
1）DPPH清除测定：取多肽溶液100 μL加入于96孔酶标板中，随后加入100 μL 0.2mmol/L DPPH乙醇溶液，振荡30s，室温避光反应30 min，在波长517 nm处测定吸光度Ai，以同样的方法同时测定100 μL多肽溶液与100 μL乙醇混合后的吸光度Aj，以及100μL 0.2mmol/L DPPH乙醇溶液与100 μL乙醇混合后的吸光度Ao。DPPH自由基清除率(DI)计算公式：
调节多肽溶液浓度至0.01～10 mg/mL，测定DPPH自由基清除率，采用SPSS24的probit回归计算IC50，即DPPH清除率达到50%时对应的浓度。
2）ABTS清除能力测定：取25 μL多肽溶液加入到96孔酶标板中，随后加入200 μL3.7 mmol/L ABTS溶液，室温避光静置6 min，在波长734 nm处测定吸光度Ai′，分别测定25μL多肽溶液与200 μL甲醇混合后的吸光度Aj′，25 μL甲醇与200 μL 3.7 mmol/L ABTS溶液混合后的吸光度Ao′。ABTS自由基清除率(AI)计算公式如下：
测定不同浓度多肽的ABTS自由基清除率，采用IBM SPSS24的概率回归算法，计算IC50。
3）ACE抑制能力测定：取马尿酰-组氨酰-亮氨酸(HHL)加入到0.05 mol/L pH8.2硼酸缓冲液（含0.3 mol/L氯化钠）配制成浓度为5 mmol/L，取0.125 mL 加入到离心管中，加入0.025 mL 4.66mmol/L ACE溶液和0.025 mL的多肽溶液，37℃反应1h，随后加入0.2 mL1 mol/L盐酸终止反应。取0.4 mL吡啶和0.2 mL 苯磺酰氯加入到溶液中，混匀1 min，冷却，410 nm测定吸光度（AS）。分别测定未加入ACE溶液和多肽溶液、未加入多肽溶液的吸光度Ab和Ac，采用如下公式计算ACE抑制率：
采用IBM SPSS24的概率回归算法，计算IC50。
芝麻多肽的活性应用，以DPPH、ABTS自由基清除能力的IC50作为抗氧化指标，ACE抑制IC50作为降血压指标，测试分析实施例1从芝麻蛋白中提取的芝麻多肽和实施例2采用人工合成的芝麻多肽SYPTECRMR的抗氧化和降血压活性，具体结果见下表1。
表1、实施例1和2所得芝麻多肽SYPTECRMR的相关活性结果
由表1可知：本发明芝麻多肽SYPTECRMR具有强抗氧化能力，尤其有极好ABTS自由基清除能力（IC50小于0.005 mg/mL），适宜在极性水溶液体系中，作为抗氧化剂使用。同时，该芝麻多肽也有一定的ACE抑制能力，可以辅助降血压药物控制病人血压。
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。
SEQUENCE LISTING
<110> 河南省农业科学院
<120> 一种芝麻多肽及其提取方法和在制备抗氧化和/或降血压药物中的应用
<130> none
<140> 2019104385468
<141> 2019-05-24
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 9
<212> PRT
<213> Sesamum indicum
<400> 1
Ser Tyr Pro Thr Glu Cys Arg Met Arg
1 5
Claims (1)
1.芝麻多肽在制备降血压药物、降血压食品、降血压营养强化剂、降血压动物饲料中的应用，其特征在于，该芝麻多肽由九个氨基酸组成，其序列为：Ser-Tyr-Pro-Thr-Glu-Cys-Arg-Met-Arg。
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