CN116751251A - 具有α-葡萄糖苷酶抑制活性的两种多肽及其应用 - Google Patents
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Abstract
本发明属于生物技术领域,特别涉及两个具有α‑葡萄糖苷酶抑制活性的多肽及其应用。本发明公开了具有α‑葡萄糖苷酶抑制活性的以下两种多肽:多肽EGEPKLP,多肽TPELKL。本发明提供的两种多肽均具有较强的α‑葡萄糖苷酶抑制活性,且具有安全无毒副作用、易于吸收等优点,可用于降血糖产品(包括降血糖药物)中。
Description
技术领域
本发明属于生物技术领域,特别涉及两个具有α-葡萄糖苷酶抑制活性的多肽及其应用。
背景技术
糖尿病是一种广泛存在的代谢紊乱疾病,根据国际糖尿病联合会的数据,2021年全球有5.37亿成年人患有糖尿病,糖尿病患者中90%为2型糖尿病。高血糖是2型糖尿病的主要特征,长期高血糖会导致各种组织的慢性损伤和功能障碍,并引起视网膜损伤、慢性肾衰竭、等并发症。因此控制血糖,尤其是餐后血糖,是2型糖尿病有效的治疗方法,而延迟碳水化合物的代谢则是控制餐后血糖的主要措施。位于小肠上皮黏膜的a-葡萄糖苷酶是参与碳水化合物代谢的关键酶,α-葡萄糖苷酶抑制剂能通过抑制α-葡萄糖苷酶的活性,延缓碳水化合物的代谢,从而控制血糖的生成速度。目前α-葡萄糖苷酶抑制剂已广泛应用于糖尿病治疗中,如阿卡波糖、伏格列波糖和米格列醇等。然而长期服用这些化学合成的药物,会导致不良反应。因此,从天然食品中寻找a-葡萄糖苷酶抑制剂,作为2型糖尿病的补充疗法已收到广泛关注。
生物活性肽是两个或两个以上的氨基酸以肽键相连的化合物,在人体内起重要生理作用,且具有溶解性好、活性高、易消化吸收,安全无毒等特点,且越来越受到人们关注。近几年有研究也发现火腿、大豆、苋菜、鸡蛋、藜麦蛋白水解肽具有α-葡萄糖苷酶抑制活性,目前已从干腌火腿、水牛奶、鹰嘴豆、黑茶等蛋白水解物中鉴定出多种具有α-葡萄糖苷酶抑制活性的抑制活性肽,如干腌火腿AEEEYPDL、LGVGG、GGLGP的IC50分别为5.58mM(5.38mg/mL)、6.36mM(2.55mg/mL)、8.71mM(3.48mg/mL);水牛奶蛋白中的YLGYLEQLLR,TKVIPYVRYL,RNAVPITPTLNR的IC50分别为470.5μM(0.596mg/mL)、498.0μM(0.623mg/mL)、503.6μM(0.681mg/mL);鹰嘴豆蛋白中的SPGAGKG和GLAR的IC50分别为1.78mg/mL和8.74mg/mL,黑茶蛋白中的CGKKFVR,AVPANLVDLNVPALLK的IC50分别为0.52mg/mL,1.03mg/mL。说明:上述IC50是针对a-葡萄糖酶抑制活性而言。
2017103998437的发明《具有降血糖功能的三肽CGP及其用途》告知:三肽CGP在5.0mg/mL时的α-葡萄糖苷酶抑制活性为35.9%。
2017104006528的发明《具有降血糖功能的三肽SPF及其用途》告知:三肽SPF在5.0mg/mL时的α-葡萄糖苷酶抑制活性为30.5%。
发明内容
本发明要解决的技术问题是提供新的具有α-葡萄糖苷酶抑制活性的两种多肽以及用途。
为解决上述技术问题,本发明提供具有α-葡萄糖苷酶抑制活性的以下两种多肽:
多肽EGEPKLP,该多肽的氨基酸序列为:Glu-Gly-Glu-Pro-Lys-Leu-Pro。
多肽TPELKL,该多肽的氨基酸序列为:Thr-Pro-Glu-Leu-Lys-Leu。
多肽EGEPKLP和TPELKL均是从香菇中性蛋白酶解液中鉴定获得;实际中,也可由多肽合成公司通过化学合成制备而得。
本发明提供的两种多肽均具有较强的α-葡萄糖苷酶抑制活性,且具有安全无毒副作用、易于吸收等优点,可用于降血糖产品(包括降血糖药物)中。
本发明的多肽EGEPKLP和TPELKL针对a-葡萄糖酶抑制活性而言,EGEPKLP的IC50为0.41mg/ml,TPELKL的IC50为0.34mg/ml。
综上,本发明的多肽具有α-葡萄糖苷酶抑制活性,且该活性未见报道,活性效应与剂量具有相关性,属于新的具有α-葡萄糖苷酶活性功能肽。
本发明的具有α-葡萄糖苷酶抑制活性的两种多肽的用法和用量如下:口服,每次约0.5g,每日3次。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细说明。
图1为不同分子量香菇酶解肽组分的α-葡萄糖苷酶抑制活性;不同的字母表示不同处理之间存在显著差异;
图2为Sephadex G-10凝胶层析法分离分子量<1kDa的组分;
图2中:A洗脱曲线;B.α-葡萄糖苷酶抑制活性;不同字母表示不同处理之间有显著差异(P<0.05)。
图3为质谱鉴定图。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此:
实例1、
1、香菇肽的制备
称取5.0g的香菇蛋白于150mL锥形瓶中,加入100mL蒸馏水搅拌混匀,调pH值至7,按酶底比3000U·g-1加入中性蛋白酶混合均匀,于45℃的温度下,放入摇床中以150r·min-1的转速酶解4h,酶解结束后,沸水浴5min使中性蛋白酶失活,冷却至室温,4000r/min离心20min,取上清液进行下一步实验。
2、超滤
将步骤1所得的上清液用1k Da、3k Da、5k Da超滤膜分离。得到的组分分别命名为UF-I(>5k Da)、UF-II(3-5kDa)、UF-III(1-3kDa)和UF-IV(<1k Da),并分别采集,测定活性。
在5mg/mL的浓度下,不同分子量香菇酶解肽组分的α-葡萄糖苷酶抑制活性如图1所示,因此可知UF-Ⅳ(<1K Da)活性较好。
3、凝胶分离
将UF-Ⅳ(<1K Da),上Sephadex G-10凝胶过滤柱(1.6×100cm),然后用超纯水以0.5mL/min的流速洗脱柱。每10min采集1根试管,在280nm处检测。收集各洗脱峰组分(图2A)并进行冻干。
共收集获得11个洗脱峰组分(F1-F11),11个洗脱峰组分(F1-F11)的α-葡萄糖苷酶抑制活性对比如图2B所示,因此可知F5组分活性最好。
4、将活性最好的F5组分进行质谱鉴定,结合分子对接进行筛选,得到2个活性肽序列----EGEPKLP和TPELKL,如图3。
而后通过化学合成法合成EGEPKLP和TPELKL后,进一步验证其活性。
实验1、本发明中所涉及的α-葡萄糖苷酶抑制活性的检测方法:
分别取50μL一定浓度的多肽溶液(0.1mol/L,pH=6.9磷酸缓冲液配置),100μL10mg/mL的α-葡萄糖苷酶溶液(用0.1mol/L,pH=6.9磷酸缓冲液配制),加入酶标板中,混匀后,25℃下放置10min。然后加入50μL 5mmol/L的PNPG(对硝基苯基-α-D-吡喃葡萄糖苷)溶液(用0.1mol/L,pH=6.9磷酸缓冲液配制),37℃下培养30min后,加入50μL0.67mol/L的Na2CO3溶液终止反应,测定405nm下吸光度。该体系称为样品。
同时设置以下3个体系:对照、样品空白、和对照空白。
对照:以50μL 0.1mol/L,pH=6.9磷酸缓冲液替代50μL的多肽溶液,其余等同于样品。
样品空白:以100μL的0.1mol/L,pH=6.9磷酸缓冲液代替100μL 10mg/mL的α-葡萄糖苷酶溶液;其余等同于样品。
对照空白:分别以相应体积量的0.1mol/L,pH=6.9磷酸缓冲液代替多肽溶液和α-葡萄糖苷酶溶液,其余等同于样品。
抑制率按下式计算。
实施例1-1、多肽EGEPKLP在0.5mg/mL的浓度下的α-葡萄糖苷酶抑制率
检测方法:将通过化学合成法获得的此多肽EGEPKLP,进行活性检测。此时多肽EGEPKLP浓度为0.5mg/mL。
A对照的2次测定平均吸光度为0.851,A对照空白的2次测定平均吸光度为0.033;A样品的2次测定吸光度分别为0.392和0.387,A样品空白的2次测定平均吸光度为0.036;
代入上式抑制率计算公式后,获得两次测定抑制率为56.5%和57.1%,平均56.8%。
结果:多肽EGEPKLP在0.5mg/mL时的α-葡萄糖苷酶抑制率为56.8%。
实施例1-2、多肽EGEPKLP在0.25mg/mL的浓度下的α-葡萄糖苷酶抑制率:
检测方法:将通过化学合成法获得的此多肽EGEPKLP,进行活性检测。此时多肽EGEPKLP浓度为0.25mg/mL。
A对照的2次测定平均吸光度为0.851,A对照空白的2次测定平均吸光度为0.033;A样品的2次测定吸光度为0.613和0.617,A样品空白的2次测定平均吸光度为0.035;
代入上式抑制率计算公式后,获得两次测定抑制率为29.3%和28.9%,平均29.1%。
结果:多肽EGEPKLP在0.25mg/mL时的α-葡萄糖苷酶抑制率为29.1%。
实施例2-1、多肽TPELKL在0.5mg/mL的浓度下的α-葡萄糖苷酶抑制率
检测方法:将通过化学合成法获得的此多肽TPELKL,进行活性检测。此时多肽TPELKL浓度为0.5mg/mL。
A对照的2次测定平均吸光度为0.851,A对照空白的2次测定平均吸光度为0.033,A样品的2次测定吸光度为0.311和0.316,A样品空白的2次测定平均吸光度为0.037;
代入上式抑制率计算公式后,获得两次测定抑制率为66.5%和65.9%,平均为66.2%。
结果:多肽TPELKL在0.5mg/mL时的α-葡萄糖苷酶抑制率为66.2%。
实施例2-2、多肽TPELKL在0.25mg/mL的浓度下的α-葡萄糖苷酶抑制率
检测方法:将通过化学合成法获得的此多肽TPELKL,进行活性检测。此时多肽TPELKL浓度为0.25mg/mL。
A对照的2次测定平均吸光度为0.851,A对照空白的2次测定平均吸光度为0.033,A样品的2次测定吸光度为0.565和0.557,A样品空白的2次测定平均吸光度为0.035;
代入上式抑制率计算公式后,获得两次测定抑制率为35.2%和36.2%,平均为35.7%。
结果:多肽TPELKL在0.25mg/mL时的α-葡萄糖苷酶抑制率为35.7%。
对比例、将表1中的香菇酶解物鉴定所得多肽,配制成浓度为0.5mg/mL的多肽溶液;然后按照上述实验方法进行α-葡萄糖苷酶抑制率测定;所得结果与本发明多肽所得结果的对比如表1所述。
表1
多肽 | α-葡萄糖苷酶抑制率(%) |
EGEPKLP(本发明) | 56.8 |
TPELKL(本发明) | 66.2 |
IDEAVPR | 13.2 |
PDDPAVVE | 15.8 |
EEPLPQ | 16.8 |
DPEKFP | 11.5 |
说明:上述IDEAVPR、PDDPAVVE、EEPLPQ、DPEKFP为发明过程中筛选而得的其余香菇肽。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形,比如不同蛋白质来源降解分离所得的不同结构及其衍生化结构。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
Claims (3)
1.具有α-葡萄糖苷酶抑制活性的多肽,其特征是为以下任一:
多肽EGEPKLP,其氨基酸序列为:Glu-Gly-Glu-Pro-Lys-Leu-Pro;
多肽TPELKL,其氨基酸序列为:Thr-Pro-Glu-Leu-Lys-Leu。
2.如权利要求1所述的具有α-葡萄糖苷酶抑制活性的多肽在制备降血糖药物中的应用。
3.根据权利要求2所述的应用,其特征是:制备作为α-葡萄糖苷酶抑制肽的药物。
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