CN113735957A - 一种新型促睡眠乳源活性肽ccl-4s及其制备方法和应用 - Google Patents
一种新型促睡眠乳源活性肽ccl-4s及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种新型促睡眠乳源活性肽CCL‑4S,所述促睡眠活性肽的氨基酸序列为Val‑Ala‑Pro‑Phe‑Pro‑Glu‑Val‑Phe‑Gly‑Lys‑Glu,分子量为1219.38。本发明以牛乳酪蛋白为原料,酶解后采用过滤、离心等基础手段除杂,以活性为导向,通过多次反向液相色谱制备及分离,得到一种具有优异促进睡眠作用的活性肽CCL‑4S。本发明促睡眠肽来源安全,无毒,对牛乳及睡眠肽产品的应用有一定指导价值。
Description
技术领域
本发明属于乳源有效成分提取技术领域,具体涉及一种新型促睡眠乳源活性肽CCL-4S及其制备方法和应用。
背景技术
失眠是一种常见的睡眠障碍,失眠患者的总睡眠时间不达标、睡眠质量不合格,且影响患者生活工作,是一种亚健康状态。睡眠障碍给患者带来多方面的不良影响,入睡时间一般超过30min;睡眠维持障碍,一般指睡眠期间觉醒次数增加,或者睡觉期间觉醒难以入睡;在睡眠的过程中,睡眠浅,多梦;次日醒来,患者头昏脑胀、精神不足、昏昏欲睡、无力等。长期失眠容易导致工作效率低,健忘,思维能力下降,产生抑郁、焦虑、精神紧张等情绪,严重的会导致悲观厌世,形成精神病、抑郁症等。
众所周知,牛奶具有促进睡眠功效,睡前喝杯热牛奶更容易入睡。牛乳酪蛋白,是许多具有潜在生物活性物质的前体,采用蛋白酶酶解,得到具有多种生物学功能的小肽,即牛乳生物活性肽。这些小肽一般由2-20个氨基酸残基组成,能够对机体的消化系统、心血管系统、免疫系统等多方面产生积极的影响,具有调节神经系统、抗高血压、抗菌、抗氧化、免疫调节等生理活性。近几年,国内外学者对牛乳肽的抗菌、抗氧化、抗高血压、免疫调节研究较多。牛乳促睡眠肽不仅功能活性强、价格便宜、安全性好,而且易于进行工业化生产。
中国发明专利CN202010387572.5公开了一种乳源活性肽CCL-3S在制备促睡眠产品中的应用,利用体外膜片钳技术从牛奶中分离鉴定出一种具有抑制神经细胞动作电位发放频率、能促进睡眠的乳源活性肽CCL-3S,该乳源活性肽氨基酸序列为HQGLPQEVLNENLLR,分子量为1759.998Da,作用于细胞后,Vm的变化率为12.56± 3.67%,与空白组比较,具有显著性抑制作用(p<0 .05),能显著抑制神经细胞动作电位发放频率。
发明内容
本发明从酪蛋白酶解物中提取分离纯化得到一种新型促睡眠乳源活性肽CCL-4S,能显著抑制神经细胞动作电位发放频率,有来源安全、促睡眠效果好、活性稳定等优点。
本发明通过以下技术方案实现:
一种新型促睡眠乳源活性肽CCL-4S,包括如SEQ ID NO:1所示的氨基酸序列,Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu(VAPFPEVFGKE),分子量为1219.38,可用于制备促睡眠产品,即功能性食品或者药品。
本发明新型促睡眠乳源活性肽CCL-4S的制备方法,包括以下步骤:
(1)原料预处理:将乳源酪蛋白酶解物原料进行浓缩,得到多肽饱和水样,采用真空喷雾干燥,得到多肽粉末;
(2)C18制备柱一次分离
将所得多肽粉末用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对促睡眠肽进行初步分离;
根据图谱中出峰时间及峰形相似度,分为6段进行接样,将这6段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性;
色谱条件:色谱柱为自装玻璃柱(20 mm×450 mm,C18填料(10μm,300 Å,MachereyNagel,France));流动相:A 泵为超纯水(含0.1% TFA),B 泵为乙腈(含0.1% TFA);流速:10mL/min;进样量:5 mL;检测波长:214 nm,;洗脱条件:10%-20%(0.01-30 min),20%-35%(30-90 min),35%-52.5%(90-110 min),52.5%-90%(110-125 min);
(3)C18制备柱二次分离
将具有最好的促睡眠活性的一段组分用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对其进行初步分离;
根据图谱中出峰时间及峰形相似度,分为3段进行接样,将这3段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性;
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min);
(4)HCLP的第三次分离
将具有最好的促睡眠活性的一段组分用超纯水配制成200mg/mL的溶液,过0.22 μm滤膜后,借助制备型高效液相色谱对其进行初步分离;
根据图谱中出峰时间及峰形相似度,分为4段进行接样,将这4段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,随后测量其体外促睡眠活性,具有最好的促睡眠活性的一段即为本发明新型促睡眠乳源活性肽CCL-4S;
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min)。
本发明具有以下积极有益效果:
本发明新型促睡眠乳源活性肽安全无毒,能更加显著地抑制神经细胞动作电位发放频率,Vm变化率达16.76±1.28,AP的抑制率达22.22±7.29,具有优异的促睡眠活性,与空白组比较,具有显著性抑制作用(p<0.01);可应用于制备促睡眠功能性食品等,并可充分利用乳产品资源,为乳源多肽的高值化利用提供了新途径。
附图说明
图1为制备液相一次分离分段接样图谱;
图2为制备液相一次分离样品AP抑制率;
图3为制备液相一次分离样品Vm的变化率;
图4为生理条件下诱发的VLPO的AP图(左)和P3对VLPO神经元诱发的AP抑制(右);
图5为制备液相二次分离分段接样图谱;
图6为制备液相二次分离样品AP抑制率;
图7为制备液相二次分离样品Vm的变化率;
图8为生理条件下诱发的VLPO的AP图(左)和P3中段对VLPO神经元诱发的AP抑制(右);
图9为第三次分离纯化HPLC图;
图10为生理条件下诱发的VLPO的AP图(左)和单体4对VLPO神经元诱发的AP抑制(右);
图11 CCL-4S氨基酸测序仪图谱。
具体实施方式
为了更好的了解本发明,下面通过具体的实施例来说明本发明的技术方案。以下实施例中所用药品和试剂均为市购,所用乳源酪蛋白酶解物为牛乳酪蛋白采用蛋白酶酶解后的牛乳多肽溶液,具体制备方法如下:酪蛋白与蒸馏水以 1:10 (w/v) 的比例混合,再将0.5%的胰蛋白酶加入酪蛋白液中水解5小时,将pH 值调至4-5,离心取上清,然后,将酶解产物(上清液)高温灭活,随后离心收集上清液,冻干,-20℃保存备用。
本发明新型促睡眠乳源活性肽,由以下方法筛选制备:
(1)原料预处理:将乳源酪蛋白酶解物原料经旋转蒸发浓缩,得到多肽饱和水样,采用真空喷雾干燥,得到多肽粉末。
(2)C18制备柱一次分离
将多肽粉末(含有大量促睡眠肽)用超纯水配制成200mg/mL的溶液,使用注射器抽取并过0.22 μm滤膜后,借助制备型高效液相色谱(LC-8A, Shimadzu)对促睡眠肽进行初步分离。
色谱条件:色谱柱为自装玻璃柱(20 mm×450 mm,C18填料(10μm,300 Å,MachereyNagel,France));流动相:A 泵为超纯水(含0.1% TFA),B 泵为乙腈(含0.1% TFA);流速:10mL/min;进样量:5 mL;检测波长:214 nm;洗脱条件:10%-20%(0.01-30 min),20%-35%(30-90 min),35%-52.5%(90-110 min),52.5%-90%(110-125 min)。
多肽粉末在制备型高效液相色谱中分离检测的图谱如图1所示,多肽常用检测波长214nm下,根据出峰时间及峰形相似度,分为6段进行接样,命名为P1、P2、P3、P4、P5、P6。将这6段组分分别收集,尽快进行浓缩(60℃旋蒸)及真空冻干,减少多肽降解。随后测量其体外促睡眠活性,对离体大鼠VLPO神经元Vm及去极化电流诱发AP的影响,结果如图2和图3所示。发现:P5样品不溶于脑脊液,P6样品量极少无法进行实验探究,且不利于后期样品富集,P1和P2样品对VLPO神经元的变化率影响较小,对去极化电流诱发AP,能抑制该神经元的兴奋性,但不具有显著性差异;P3具有较好的抑制平均AP诱发的个数,P3作用后Vm的具有显著性变化。而P4能显著性的改变作用后VLPO神经元的Vm,但对诱发的神经元,不具有显著的抑制。因此,P3具有较好的抑制VLPO神经元的功效,对P3段活性具体结果分析如下。
表1显示,空白对照组的Vm变化为5.73±1.22%,P3作用VLPO神经元后,Vm的变化率为21.20±4.65%,与空白组比较,牛乳促睡眠肽对VLPO神经元具有显著性差异。以去极化电流诱发AP的个数评估P3对腹外侧视前区神经元兴奋性的影响,见图4。空白对照组经去极化电流刺激后,其诱发的AP的抑制率为6.89±2.2.7%,P3作用5min后,能显著诱发的AP平均个数,抑制率为25.00±6.35%,牛乳促睡眠肽能显著抑制诱发的AP。结果表明,在不影响腹外侧视前区神经元正常生理特性下,P3能显著抑制VLPO神经元的兴奋性(p<0.01)。
表1 P3对VLPO神经元兴奋性的影响(n=16)
注:与正常对照组比较,*p<0.05,**p<0.01。
(3)C18制备柱二次分离
根据以上研究发现P3具有较好的促睡眠活性,将P3组分用超纯水配制成200mg/mL的溶液,使用注射器抽取并过0.22 μm滤膜后,借助制备型高效液相色谱(LC-8A,Shimadzu)对其进行初步分离。P3组分在制备型高效液相色谱中分离检测的图谱如图5所示,多肽常用检测波长214nm下,根据出峰时间及峰形,将P3组分分为前中后三段,浓缩冻干,继续评价体外促睡眠活性。
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48min)。
见图6和图7,牛乳促睡眠肽P3前、中、后段均有较好的促睡眠活性,均具有较好的抑制平均AP诱发的个数,作用后Vm均具有显著性变化。三者抑制VLPO神经元效果对比发现,P3中段的抑制效果最好,而P3前段和P3后段的抑制效果类似。P3中段活性具体分析如下:
空白对照组的Vm变化为3.97±1.22%,P3中段作用细胞后,Vm的变化率为22.94±3.65%,与空白组比较,牛乳促睡眠肽P3中段有显著性抑制作用(p<0.01)。以去极化电流诱发AP的个数评估P3中段对VLPO神经元兴奋性的影响,见图8。空白对照组经去极化电流刺激后,其诱发的AP的抑制率为6.06±4.62%,P3中段作用5min后,能显著诱发的AP平均个数,抑制率为25.00±7.51%,两组间有显著性差异(p<0.01)。结果表明,在不影响VLPO神经元正常生理特性下,P3中段具有较好的抑制平均AP诱发的个数,P3中段作用后Vm的具有显著性变化。
表2 P3中段对VLPO神经元兴奋性的影响(n=14)
注:与正常对照组比较,*p<0.05,**p<0.01。
(4)HCLP的第三次分离
根据以上研究发现P3中段具有较好的促睡眠活性,用以上同样的方法将P3中段分为4个单峰,浓缩冻干,进行体外促睡眠活性探索,见图9,发现单体4(CCL-4S)具有良好的促睡眠活性。
色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm,280 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48 min)。
表3显示,空白对照组的Vm变化为2.59±1.09%,单体4作用细胞后,Vm的变化率为16.76±1.28%,与空白组比较,牛乳促睡眠肽单体4有显著性抑制作用(p<0.01)。以去极化电流诱发AP的个数评估单体4对VLPO神经元兴奋性的影响,见图10。空白对照组经去极化电流刺激后,其诱发的AP的抑制率为2.22±1.47%,单体4作用5min后,能显著诱发的AP平均个数,抑制率为22.22±7.29%,两组间有显著性差异(p<0.01)。结果表明,在不影响VLPO神经元正常生理特性下,单体4具有较好的抑制平均AP诱发的个数,单体4作用后Vm的具有显著性变化。
表3 单体4对VLPO神经元兴奋性的影响(n=9)
注:与正常对照组比较,*p<0.05,**p<0.01。
(5)促睡眠乳源活性肽的一级结构鉴定
利用氨基酸测序仪(PPSQ)测定CCL-4S多肽段N端绝对序列,见图11,通过比对Uniprot数据库中牛乳酪蛋白蛋白序列,确定多肽的序列为Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys-Glu(VAPFPEVFGKE),分子量为1219.38,来源于牛乳酪蛋白中的Alpha-S1-酪蛋白,这是首次从食物源天然产物中分离出此序列促睡眠肽。
体外促睡眠活性的测定运用膜片钳技术。首先制作下丘脑切片,取出生5周左右的大鼠,体重约100g-150g,急性分离其下丘脑,将下丘脑切片为400μm,接着制备微电极,将切片迅速放入充满95%O2和5%的人工脑脊液中,在33℃中孵育30min,拿出孵育槽,在室温下放置20min。采用外径为1.4mm的有芯硬质玻璃毛坯,抛光后尖端拉制为直径约2μm,充满电极内液。其次将微电极与下丘脑切片的VLPO神经元进行封接,用5× 的物镜观察下丘脑的结构,找到腹外侧视前区,然后用40× 浸水镜观察单个腹外侧视前区神经元的形态并利用电极进行封接,当电极尖端与细胞间形成大于1GΩ封接后成功后,在封接窗口观察到细胞出现电容充放电后,即形成全细胞模式。首先记录一段自发放电作为实验对照,通常2-3min,膜电位50mV,稳定后给药。然后灌流含牛乳多肽的灌流液,以观察神经元放电形式的变化,通过评价Vm的变化率与去极化电流诱发AP的个数评估VLPO神经元兴奋性抑制率的影响,从而分析多肽的体外促睡眠活性。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,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。
序列表
<110> 华南农业大学
<120> 一种新型促睡眠乳源活性肽CCL-4S及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11
<212> PRT
<213> 多肽(polypeptide)
<400> 1
Val Ala Pro Phe Pro Glu Val Phe Gly Lys Glu
1 5 10
Claims (10)
1.一种新型促睡眠乳源活性肽CCL-4S,其特征在于:包括如SEQ ID NO:1所示的氨基酸序列。
2.权利要求1所述新型促睡眠乳源活性肽CCL-4S在制备促睡眠产品中的应用。
3.一种促睡眠产品,其特征在于:其原料中包括权利要求1所述的新型促睡眠乳源活性肽CCL-4S。
4.根据权利要求3所述的促睡眠产品,其特征在于:所述促睡眠产品为食品或者药品。
5.权利要求1所述新型促睡眠乳源活性肽CCL-4S的制备方法,其特征在于,包括以下步骤:
(1)将原料乳源酪蛋白酶解物浓缩,得到多肽饱和水样,真空喷雾干燥,得到多肽粉末;
(2)将所得多肽粉末用配制成水溶液,过滤后将滤液借助制备型高效液相色谱进行初步分离;根据图谱中出峰时间及峰形相似度,分为6段进行接样,将组分分别收集,进行浓缩并真空冻干,分别测量其体外促睡眠活性;
(3)将上一步骤所得具有最好的促睡眠活性的一段组分配制成水溶液,过滤后将滤液借助制备型高效液相色谱对其进行初步分离;根据图谱中出峰时间及峰形相似度,分为3段进行接样,将组分分别收集,进行浓缩并真空冻干,分别测量其体外促睡眠活性;
(4)将上一步骤所得具有最好的促睡眠活性的一段组分配制成水溶液,过滤后将滤液借助制备型高效液相色谱对其进行初步分离;根据图谱中出峰时间及峰形相似度,分为4段进行接样,将组分分别收集,进行浓缩并真空冻干,分别测量其体外促睡眠活性,具有最好的促睡眠活性的一段即为本发明新型促睡眠乳源活性肽CCL-4S。
6.根据权利要求5所述的制备方法,其特征在于:步骤(2)中的色谱条件:色谱柱为自装玻璃柱(20 mm×450 mm,C18填料(10μm,300 Å,Macherey Nagel,France));流动相:A 泵为超纯水(含0.1% TFA),B 泵为乙腈(含0.1% TFA);流速:10 mL/min;进样量:5 mL;检测波长:214 nm;洗脱条件:10%-20%(0.01-30 min),20%-35%(30-90 min),35%-52.5%(90-110min),52.5%-90%(110-125 min)。
7.根据权利要求5所述的制备方法,其特征在于:步骤(3)中的色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48 min)。
8.根据权利要求5所述的制备方法,其特征在于:步骤(4)中的色谱条件:色谱柱为Welch C18 柱子(4.6 mm×250 mm,5 μm,Ultimate);流动相:A泵为超纯水(含0.1% TFA),B泵为乙腈(含0.1% TFA);进样量:20 µL;流速:1 mL/min;检测波长:214 nm;洗脱条件:10%-20%(0.01-10 min),20%-30%(10-40 min),30%-90%(40-48 min)。
9.根据权利要求5所述的制备方法,其特征在于:所述的浓缩为60℃旋蒸,所述的配制成水溶液为用超纯水配置成200mg/mL的溶液;所述的过滤为过0.22 μm滤膜。
10.根据权利要求5所述的制备方法,其特征在于:所用乳源酪蛋白酶解物为牛乳酪蛋白采用蛋白酶酶解后的牛乳多肽溶液,具体制备方法如下:将适量胰蛋白酶加入酪蛋白中水解2-7小时,再将pH 值调至2-7,离心取上清,然后,将所得上清液高温灭活,随后离心收集上清液,冻干,-20℃保存备用。
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